Assessing endothelial function and providing calibrated ufmd data using a blood pressure cuff

ABSTRACT

Methods and apparatus are provided for assessing endothelial function in a mammal. In certain embodiments the methods involve using a cuff to apply pressure to an artery in a subject to determine a plurality of baseline values for a parameter related to endothelial function as a function of applied pressure (P m ); b) applying a stimulus to the subject; and applying external pressure P m  to the artery to determine a plurality of stimulus-effected values for the parameter related to endothelial function as a function of applied pressure (P m ); where the baseline values are determined from measurements made when said mammal is not substantially effected by said stimulus and differences in said baseline values and said stimulus-effected values provide a measure of endothelial function in said mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Ser. No. 15/236,250, filed onAug. 12, 2016, now U.S. Pat. No: 9,737,217, which claims priority to andbenefit of U.S. Ser. No. 62/205,470, filed on Aug. 14, 2015, both ofwhich are incorporated herein by reference in their entirety for allpurposes.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under Contract No.DE-ACO2-05CH11231, awarded by the U.S. Department of Energy. Thegovernment has certain rights in the invention.

BACKGROUND

Impairment of arterial endothelial function is an early event inatherosclerosis and correlates with all of the major risk factors forcardiovascular disease (CVD). The most widely employed noninvasivemeasure of endothelial function involves brachial artery (BA) diametermeasurement using ultrasound imaging before and after several minutes ofblood flow occlusion (Celermajer et al. (1992) The Lancet, 340:1111-1115). The change in arterial diameter is a measure offlow-mediated vasodilation (FMD). This peripheral measurement correlatesstrongly with coronary artery endothelial function, a fact that stronglysupports its clinical value. However, the high between-laboratoryvariability of results and cost of instrumentation render this techniqueunsuitable for routine clinical use.

Endothelial function is both acutely and chronically affected bylifestyle factors that influence CVD risk (Brunner et al. (2005) J.Hypertens., 23: 233-246). Consequently, measures of endothelial functionare useful in monitoring response to medication, dietary changes andexercise regimens. Unfortunately, very little work has focused ondetermining the clinical value of endothelial function measurements forindividual patients or on developing measurement methods suitable forroutine or continuous monitoring of endothelial function. There arecompelling reasons to believe that knowledge of acute variation inendothelial function in an individual is important. Since NO released bythe endothelium is a potent inhibitor of leukocyte and monocyte adhesionto the endothelial cell surface, and since adhesion of these cells iswidely believed to be a necessary initiating event in atherogenesis(Deanfield et al. (2005) J. Hypertens., 23: 7-17), it is reasonable toinfer that the proportion of time that the endothelium is dysfunctionalconstitutes an important indicator of disease risk. This is therationale for the development of techniques that are simple and cheapenough to enable regular or continuous measurement of endothelialfunction.

The two FDA-approved commercially available systems for measuringendothelial function perform measurements that are based on the flow andpulse pressure in resistance vessels (rather than in conduit arteries).The Endo-PAT2000 system from Itamar Medical analyzes the pulse amplitudein the finger before and after application on endothelial stimulus.While about 46% of the observed changes in pulse amplitude are blockedby NO synthase inhibitors, mechanisms other than those mediated by NOsignificantly contribute towards the response (Nohria et al. (2006) JAppl Physiol, 101(2): 545-548). This is most probably a consequence ofthe different mechanisms involved in arterial andarteriolar/microvascular vasodilation. Also, the measurement is made invessels that experience ischemia and the many non-NO-mediatedvasodilatory processes that occur under ischemic conditions. It isclinically preferable to perform measurements on arteries such as thebrachial artery, the endothelial response of which is highly correlatedwith that of the coronary arteries (r=0.78, p<0.001, Takase et al.(1998) Am. J Cardiol., 82(12): 1535-1539). In addition, a review ofclose to 2,500 studies found that brachial and coronary artery EF havesimilar power to predict serious cardiovascular events over a follow-upperiod of 1-92 months (Lerman and Zeiher (2005) Circulation, 111(3):363-368). The authors of the review assert that “the similar power ofcoronary and peripheral endothelial dysfunction to predictcardiovascular events and the observation that the cardiovascular eventsmay occur remotely from the site in which the endothelial dysfunctionwas detected underscore the systemic nature of endothelial dysfunctionand its pivotal role in prediction of cardiovascular events.” It is notcurrently possible to make such strong statements regarding thesignificance of microvascular endothelial function.

Two large (>1800 subject) cross-sectional studies found an associationbetween EndoPAT measurements and accepted cardiovascular risk factors(Palmisano et al. (2011) Hypertension, 57(3): 390-396; Schnabel et al.(2011) Circulation: Cardiovascular Imaging, 4(4): 371-380). However, thecorrelations between EndoPAT and FMD were low in the respective studies:r=0.094 (N=1843), and r=0.19 (N=5000). Correlations decreased furtherwhen adjusted for age and sex. Also, some of the results suggest theinfluence of potentially serious confounding factors. For example, whileit is well known that endothelial function tends to decrease with age,older subjects exhibited better endothelial response according toEndo-PAT (Hamburg et al. (2008) Circulation, 117(19): 2467-2474).

A second approved device is the Vendys system developed by Endothelix,Inc. of Houston Tex. This system measures the cutaneous reactivehyperemic response using hand skin temperature measurement during twominutes of brachial artery occlusion and ensuing RH. During occlusion,skin temperature drops in the distal hand. As blood flow is restored,the temperature increases. Studies indicate that the recovery of skintemperature is slowed in subjects having higher Framingham risk scoresand other metrics of CVD and CVD risk. Interestingly, substantialtemperature changes are also observed in the contralateral hand thatexperiences no reactive hyperemic episode. This suggests significantneural involvement in the response. For this reason and the results ofWong et al. (2003)J. Appl. Physiol., 95: 504-510 it is reasonable topredict that this response cannot be blocked by NOS inhibitors.

There is no doubt that these systems provide clinical value and canidentify patients with pooled cardiovascular risk factors. However, itis not clear that these systems can do this better than paper-basedscoring methods such as the Framingham risk in general populations. Itis also highly probable that sympathetic nervous activation is asignificant confounding factor in endothelial function measurementsbased on arteriolar and microvascular responses.

Much stronger evidence exists that peripheral artery endothelialfunction provides more than simply a correlate of CVD risk factors. Fewclinicians would disagree with the statement that evaluation of EF inconduit arteries has more proven clinical value.

SUMMARY

Conventional flow mediated dilation (FMD) studies measure arterialdiameter before and after the application of an endothelial stimulus. Inthe methods and apparatus described herein the cross sectional area ofthe artery rather than the diameter is measured. Rather than employingB-mode ultrasound to image the arterial lumen, a simple inexpensiveblood pressure cuff is utilized.

Throughout this document, the term “area” measurement is not necessarilytaken to mean an actual calibrated measurement of area, but rather aquantity proportional to, or representative of, arterial luminal area.In most embodiments, this area measure is obtained from the amplitude ofthe pulse waveform, sensed as pressure changes in a cuff. The reason acalibrated measure of area is not required for the methods taught here,is that key calculations involve the ratio of “areas” measured beforeand after application of a stimulus, and these ratios are independent ofabsolute calibration factors. Measures such as the ratio of post- topre-stimulus pressure waveform amplitudes constitute “measured areas”for this purpose.

Various embodiments contemplated herein may include, but need not belimited to, one or more of the following:

Embodiment 1: A method of assessing endothelial function in a mammal,said method comprising: a) applying external pressure P_(m) to an arteryfor a period of time T_(m), and determining, over the course of one ormore cardiac cycles in said period T_(m), changes in pressure in a cuffresulting from cardiac activity of said mammal, or an artificiallyinduced arterial pulse, to determine values for a baseline parameterrelated to endothelial function in said mammal, where said baselineparameter is determined for at least three different external pressurepoints (P_(m)) by varying the external pressure P_(m) during period oftime T_(m), and/or by determining said baseline parameter over aplurality of time periods T_(m) having different external pressurepoints (P_(m)), to provide a plurality of baseline values for saidparameter related to endothelial function as a function of appliedpressure (P_(m)); b) applying a stimulus to said mammal; and c) applyingexternal pressure P_(m) to an said artery for a period of time T, anddetermining over the course of one or more cardiac cycles in said periodT_(m), changes in pressure in said cuff resulting from cardiac activityof said mammal, or an artificially induced arterial pulse, to determinevalues for a stimulus-effected parameter related to endothelial functionin said mammal, where said stimulus-effected parameter is determined forat least three different external pressure points (P_(m)) by varying theexternal pressure P_(m) during period of time T_(m), and/or bydetermining said parameter over a plurality of time periods T_(m) havingdifferent external pressure points (P_(m)), to provide a plurality ofstimulus-effected values for said parameter related to endothelialfunction as a function of applied pressure (P_(m)); wherein saidbaseline values are determined from measurements made when said mammalis not substantially effected by said stimulus and differences in saidbaseline values and said stimulus-effected values provide a measure ofendothelial function in said mammal.

Embodiment 2: The method of embodiment 1, wherein said method furthercomprises: determining the area (e.g., a measure proportional to thearea) of the arterial lumen calculated from the baseline pressuremeasurements as a function of transmural pressure P_(tm), wheretransmural pressure is determined as the difference between the baselinemeasured pressures and the external cuff pressure P_(m) and fittingthese data with a first non-linear model to provide a first functiondescribing baseline arterial lumen area as function of transmuralpressure; determining the area (e.g., a measure proportional to thearea) of the arterial lumen calculated from the stimulus-effectedpressure measurements as a function of transmural pressure P_(tm), wheretransmural pressure is determined as the difference between the measuredstimulus-effected pressures and the external cuff pressure P_(m) andfitting these data with a second non-linear model to provide a secondfunction describing stimulus-effected arterial lumen area as function oftransmural pressure; using said first function to calculate baselinearterial lumen area (A_(b)) (e.g., a measure proportional to the areaA_(b)) at a transmural pressure substantially equal to the systolicblood pressure; using said second function to calculatestimulus-effected arterial lumen area (A_(r))) (e.g., a measureproportional to the area A_(r)) at a transmural pressure substantiallyequal to the systolic blood pressure; and determining the equivalentultrasound-based FMD (uFMD) measure where uFMD is proportional to thesquare root of the ratio A_(r)/A_(b).

Embodiment 3: The method of embodiment 2, comprising outputting saidmeasure to a display, printer, or computer readable medium.

Embodiment 4: The method according to any one of embodiments 2-3,wherein the equivalent ultrasound-based FMD is given as

uFMD%=[√{square root over ((A _(r) /A _(b))}−1]×100

Embodiment 5: The method according to any one of embodiments 2-4,wherein said first non-linear model and/or said second non-linear modelare the same type of function.

Embodiment 6: The method according to any one of embodiments 2-5,wherein said first non-linear model and/or said second non-linear modelare selected from the group consisting of a 2 parameter model, a 3parameter model, a four parameter model, a 5 parameter model, and a sixparameter model.

Embodiment 7: The method according to any one of embodiments 2-5,wherein said first non-linear model and said second non-linear model arethree parameter models.

Embodiment 8: The method of embodiment 7, wherein said first non-linearmodel and said second non-linear model are arctangent models.

Embodiment 9: The method according to any one of embodiments 1-8,wherein baseline values and stimulus effected values are determined forthe same limb or region.

Embodiment 10: The method according to any one of embodiments 1-8,wherein baseline values are determined for a limb or regioncontralateral to a limb or region used for determining the stimuluseffected values.

Embodiment 11: The method of embodiment 10, wherein said contralaterallimb or region is used for monitoring blood pressure during measurement.

Embodiment 12: The method of embodiment 11, wherein the monitored bloodpressure is used to adjust the measurement pressure if blood pressurechanges during the measurement.

Embodiment 13: The method according to any one of embodiments 1-12,wherein said applying external pressure P_(m) to an said artery for aperiod of time T_(m) comprises applying a substantially constantexternal pressure P_(m) for a period of time T_(m).

Embodiment 14: The method according to any one of embodiments 1-12,wherein said applying external pressure P_(m) to an said artery for aperiod of time T_(m), comprises applying a continuously varying externalpressure P_(m) for a period of time T_(m).

Embodiment 15: The method according to any one of embodiments 1-14,wherein step (a) and/or step (b) comprises determining said values for abaseline parameter and/or said values for a stimulus-effected parameterfor at least 3 different pressures (P_(m)), or for at least 4 differentpressures (P_(m)), or for at least 5 different pressures (P_(m)), or forat least 6 different pressures (P_(m)), or for at least 7 differentpressures (P_(m)), or for at least 8 different pressures (P_(m)), or forat least 9 different pressures (P_(m)), or for at least 10 differentpressures (P_(m)), or during a continuous pressure variation.

Embodiment 16: The method according to any one of embodiments 1-15,wherein said external pressure is less than the mean arterial pressure.

Embodiment 17: The method according to any one of embodiments 1-15,wherein said external pressure is less than the mean diastolic pressure.

Embodiment 18: The method according to any one of embodiments 1-17,wherein said external pressure (P_(m)) ranges from about 20 mmHg up toabout 80 mmHg.

Embodiment 19: The method according to any one of embodiments 1-18,wherein the external pressure points (P_(m)) used to determine saidbaseline values are substantially the same as the external pressurepoints (PM) used to determine the stimulus-effected values.

Embodiment 20: The method according to any one of embodiments 1-18,wherein the external pressure points (P_(m)) used to determine saidbaseline values are different than the external pressure points (PM)used to determine the stimulus-effected values.

Embodiment 21: The method according to any one of embodiments1-20,wherein said baseline parameter is determined for at least threedifferent external pressure points (P_(m)) by varying the externalpressure P_(m) during period of time T_(m).

Embodiment 22: The method according to any one of embodiments 1-21,wherein said baseline parameter is determined for at least threedifferent external pressure points (P_(m)) by determining said baselineparameter over a plurality of time periods T_(m) having differentexternal pressure points (P_(m)).

Embodiment 23: The method according to any one of embodiments 1-22,wherein said stimulus-effected parameter is determined for at leastthree different external pressure points (P_(m)) by varying the externalpressure P_(m) during period of time T_(m).

Embodiment 24: The method according to any one of embodiments 1-23,wherein said stimulus-effected parameter is determined for at leastthree different external pressure points (P_(m)) by determining saidparameter over a plurality of time periods T_(m) having differentexternal pressure points (P_(m)).

Embodiment 25: The method according to any one of embodiments 1-24,wherein the step (a) is repeated at least twice, or at least 3 times, orat least 5 times, or at least about 10 times.

Embodiment 26: The method according to any one of embodiments 1-25,wherein the step (c) is repeated at least twice, or at least 3 times, orat least 5 times, or at least about 10 times.

Embodiment 27: The method according to any one of embodiments 1-26,wherein the duration of said time interval(s) (T_(m)) used to determinesaid baseline values and/or said stimulus-effected values ranges fromabout 1 sec, or from about 2 sec, or from about 3 sec, or from about 4sec, or from about 5 sec, or from about 6 sec, or from about 7 sec, orfrom about 8 sec, or from about 9 sec, or from about 10 sec, or fromabout 15 sec up to about 20 sec, or up to about 30 sec or up to about 40sec or up to about 50 sec, or up to about 1 min, or up to about 2 min,or up to about 3 min, or up to about 4 min, or up to about 5 min, or upto about 6 min, or up to about 7 min, or up to about 8 min, or up toabout 9 min, or up to about 10 min, or up to about 15 min, or up toabout 20 min, or up to about 25 min, or up to about 30 min.

Embodiment 28: The method according to any one of embodiments 1-26,wherein the duration of said time interval(s) (T_(m)) used to determinesaid baseline values and/or said stimulus-effected values is about 30seconds.

Embodiment 29: The method according to any one of embodiments 1-28,wherein there are a plurality of measurement periods (T_(m)) with awaiting period of at least 30 seconds between cuff inflations.

Embodiment 30: The method according to any one of embodiments 1-29,wherein during said time interval(s) said external pressure ismaintained substantially constant and held at different levels indifferent time intervals.

Embodiment 31: The method according to any one of embodiments 1-29,wherein said pressure is adjusted to different levels during a singletime period (T_(m)).

Embodiment 32: The method according to any one of embodiments 1-31,wherein said establishing baseline values and/or determiningstimulus-effected values comprises determining values for changes inpressure resulting from an artificially induced arterial pulse.

Embodiment 33: The method according to any one of embodiments 1-31,wherein said establishing baseline values and/or determiningstimulus-effected values comprises determining values for changes inpressure resulting from cardiac activity of said mammal.

Embodiment 34: The method according to any one of embodiments 1-33,wherein said cuff is disposed around an arm or leg of said mammal.

Embodiment 35: The method according to any one of embodiments 1-34,wherein said cuff is pressurized by a gas or gas mixture.

Embodiment 36: The method according to any one of embodiments 1-34,wherein said cuff is pressurized by a liquid or gel.

Embodiment 37: The method according to any one of embodiments 1-36,wherein said mammal is a human.

Embodiment 38: The method according to any one of embodiments 1-36,wherein said mammal is a non-human mammal.

Embodiment 39: The method according to any one of embodiments 1-38,wherein said external pressure is maintained by a system that monitorsand adjusts the pressure in said cuff and whose response time issufficiently slow so that the changes in pressure resulting from saidcardiac activity are not substantially attenuated by said system.

Embodiment 40: The method of embodiment 39, wherein said response timeis sufficiently slow so that said pressure changes resulting from saidcardiac activity are attenuated by less than 10%.

Embodiment 41: The method according to any one of embodiments 1-40,wherein said external pressure is maintained by setting the pressure insaid cuff to a value and not altering external pressure applied to saidcuff during the measurements of pressure variations due to said cardiacactivity.

Embodiment 42: The method according to any one of embodiments 1-41,wherein applying the pressure to the artery comprises applying a localpressure that does not substantially affect other blood vessels in asame limb as the artery.

Embodiment 43: The method according to any one of embodiments 1-41,wherein applying the external pressure to the artery comprises applyinga pressure that affects an entire cross-section of a limb including theartery.

Embodiment 44: The method according to any one of embodiments 1-43,wherein said external pressure is equivalent to or below the averagediastolic pressure measured for said subject.

Embodiment 45: The method of embodiment 44, wherein said substantiallyexternal pressure is below the average diastolic pressure measured forsaid subject or below an expected diastolic pressure for said subject.

Embodiment 46: The method according to any one of embodiments 1-45,wherein said substantially constant external pressure is set topredetermined pressures.

Embodiment 47: The method according to any one of embodiments 1-46,wherein the baseline values are determined before applying the stimulus.

Embodiment 48: The method according to any one of embodiments 1-47,wherein the determining stimulus-effected values is performed at leastabout 30 seconds after stimulus, or at least about 45 seconds after saidstimulus, or at least about 6o seconds after said stimulus, or at leastabout 90 seconds after said stimulus.

Embodiment 49: The method according to any one of embodiments 1-48,wherein the determining stimulus-effected values is performed up toabout 5 minutes after said stimulus, or up to about 4 minutes, or up toabout 3 minutes, or up to about 2 minutes or up to about 90 secondsafter said stimulus.

Embodiment 50: The method according to any one of embodiments 1-46,wherein the baseline values are determined after applying the stimulus.

Embodiment 51: The method according to any one of embodiments 1-50,wherein said determining, over the course of one or more cardiac cycles,changes in pressure in said cuff resulting from cardiac activity of saidmammal comprises determining the pressure in said cuff as a function oftime.

Embodiment 52: The method of embodiment 51, wherein said determiningcomprises integrating the value of a pressure change over time(calculating the area under a pressure/time curve) for one or for aplurality of cardiac cycles to determine an integrated pressure value.

Embodiment 53: The method according to any one of embodiments 51-52,wherein said determining comprises determining the maximum, or a certainpercentile rank of the derivative of the pressure versus time wave formon the rising edge of a pressure pulse for one or for a plurality ofcardiac cycles to determine a compliance value.

Embodiment 54: The method according to any one of embodiments 52-53,wherein said integrated pressure value and/or said compliance value isaveraged over a plurality of cardiac cycles.

Embodiment 55: The method according to any one of embodiments 52-53,wherein said integrated pressure value and/or said compliance value isdetermined for a single cardiac cycle.

Embodiment 56: The method of embodiment 55, wherein said single cardiaccycle is a cardiac cycle selected for the maximum change in said valuein a plurality of cardiac cycles.

Embodiment 57: The method of embodiment 55, wherein said single cardiaccycle is a cardiac cycle selected for the maximum change in said valuebetween a baseline measurement and a stimulus-effected measurement.

Embodiment 58: The method of embodiment 51, wherein said determining,over the course of one or more cardiac cycles, changes in pressure insaid cuff resulting from cardiac activity of said mammal comprisesdetermining the pressure in said cuff as a function of time to provide acardiac pulse waveform.

Embodiment 59: The method of embodiment 58, wherein said determining,over the course of one or more cardiac cycles, changes in pressure insaid cuff resulting from cardiac activity of said mammal comprisesdetermining the pressure in said cuff as a function of time to provide acardiac pulse waveform while the cuff inflates and/or while said cuffdeflates.

Embodiment 60: The method of embodiment 59, wherein said determining iswhile said cuff inflates and/or deflates as a rate of about 3 to 6mmHg/s.

Embodiment 61: The method according to any one of embodiments 59-60,wherein the cardiac pulse waveforms are compared between baseline andpost-stimulus conditions by direct comparison of pulse characteristicsat corresponding pressure levels.

Embodiment 62: The method according to any one of embodiments 59-60,wherein the cardiac pulse waveforms are compared between baseline andpost-stimulus conditions by first fitting models to the set of baselinecardiac pulse waver forms and to the set of post-stimulus cardiac pulsewaveforms and comparing parameters generated by the two models.

Embodiment 63: The method according to any one of embodiments 59-62,wherein blood pressure and cFMD are measured simultaneously.

Embodiment 64: The method according to any one of embodiments 59-63,wherein said cuff is inflated to a pressure in excess of the peak of thecardiac pulse waveform (oscillometric waveform).

Embodiment 65: The method according to any one of embodiments 59-64,wherein the systolic BP and MAP are measured and the diastolic bloodpressure (DBP) is calculated using an oscillometric analysis.

Embodiment 66: The method according to any one of embodiments 59-65,wherein the systolic BP and MAP are measured and the DBP is determinedby analyzing

Korotkoff sounds obtained using an audio sensor (e.g., stethoscope,phonocadiogram) or ultrasound probe.

Embodiment 67: The method according to any one of embodiments 59-66,wherein measurements to calculate cFMD and/or MAP, and/or DBP areobtained during cuff inflation.

Embodiment 68: The method according to any one of embodiments 59-67,wherein measurements to calculate cFMD and/or MAP, and/or DBP areobtained during cuff deflation.

Embodiment 69: The method according to any one of embodiments 59-68,wherein systolic BP and/or diastolic BP is determined and for cFMDdetermination, comparison of pulse waveform characteristics betweenbaseline and post-stimulus intervals at corresponding pressures is acomparison made where the corresponding pressures are transmuralpressures calculated using the determined systolic and/or diastolic BPvalues.

Embodiment 70: The method of embodiment 62, wherein comparison of pulsewaveform characteristics between baseline and post-stimulus intervals atcorresponding pressures is a comparison made based on the transmuralpressure determined by the models.

Embodiment 71: The method according to any one of embodiments 59-70,wherein: measurements are made of the same mammal at a plurality ofdifferent times; and measurement(s) made at earlier times in saidplurality are used to determine the tone sate of the vessel at thecurrent measurement time.

Embodiment 72: The method of embodiment 71, wherein the degree ofdilation (tone state) at baseline is used to adjust the cFMD and BPmeasurement s to the current vessel tone state.

Embodiment 73: The method according to any one of embodiments 71-72,wherein the maximal dilated state is measured by stimulating the vesselwith an NO donor such as nitroglycerin.

Embodiment 74: The method according to any one of embodiments 71-73,wherein a constricted state is measured by stimulating the vessel with avasoconstrictive sub stance.

Embodiment 75: The method according to any one of embodiments 71-72,wherein which the dilatory and constrictory potential of the vessel andtone state are inferred by measurements obtained with the cuff elevatedat different vertical levels with respect to the heart.

Embodiment 76: The method according to any one of embodiments 71-75,wherein a model describing the dependence of the oscillometriccharacteristic on vessel tone is used to define thresholds foroscillometric determination of DBP.

Embodiment 77: The method according to any one of embodiments 1-76,wherein during said time intervals pressure is applied to said cuffusing a control feedback system to adjust a pump or other pressuresource and/or a proportional release valve to alter the appliedpressure.

Embodiment 78: The method according to any one of embodiments 1-76,wherein during said time intervals said pressure is periodicallyadjusted using an on-off control system.

Embodiment 79: The method according to any one of embodiments 1-76,wherein during said time intervals adjustment of said pressure is by apump and a proportional valve that operate simultaneously to maintainpressure where pump remains on while the valve is adjusted to maintain aconstant pressure, this system being characterized by a time constantsuch that a cardiac pulse signal is not appreciably attenuated by theservo mechanism.

Embodiment 80: The method according to any one of embodiments 1-79,wherein applying the stimulus comprises restricting flow of blood to thelimb by occlusion of a blood vessel.

Embodiment 81: The method of embodiment 80, wherein restricting the flowof blood is accomplished using a cuff and/or a tourniquet.

Embodiment 82: The method of embodiment 80, wherein restricting the flowof blood and applying the pressure on the artery are performed usingseparate cuffs.

Embodiment 83: The method of embodiment 80, wherein the same cuff isused to occlude the blood vessel and to apply the pressure on theartery.

Embodiment 84: The method according to any one of embodiments 82-83,wherein restricting flow of blood through the artery comprises inflatingthe restricting cuff to a pressure at least 10 mm Hg above measuredsystolic blood pressure for said mammal.

Embodiment 85: The method according to any one of embodiments 80-84,wherein restricting flow of blood through the artery comprisesrestricting for at least 1 minute.

Embodiment 86: The method according to any one of embodiments 1-57,wherein applying the stimulus comprises administering a drug to saidmammal.

Embodiment 87: The method of embodiment 86, wherein said drug is not anNO agonist.

Embodiment 88: The method according to any one of embodiments 86-87,wherein said drug is a β-adrenergic agonist.

Embodiment 89: The method of embodiment 86, wherein said drug is an NOdonor.

Embodiment 90: The method of embodiment 89, wherein said drug comprisesnitroglycerin or sodium nitroprusside.

Embodiment 91: The method according to any one of embodiments 1-57,wherein said stimulus does not comprise occlusion of an artery and/ordoes not comprise administration of a drug.

Embodiment 92: The method of embodiment 91, wherein said stimuluscomprises low intensity ultrasound.

Embodiment 93: The method of embodiment 91, wherein said stimuluscomprises acoustic/mechanical tissue vibration.

Embodiment 94: The method according to any one of embodiments 1-91,wherein said measure of endothelial function, in the context ofdifferential diagnosis, is an indicator of a cardiovascular pathology.

Embodiment 95: The method of embodiment 94, wherein said cardiovascularpathology is a condition selected from the group consisting ofAtherosclerosis, hypercholesterolemia, high LDL-C, low HDL-C, highlipoprotein (a), small dense LDL-C, oxidized LDL-C, hypertension, highhomocysteine, aging, vasculitis, preeclampsia, metabolic syndrome,variant angina, diabetes, active smoking, passive smoking,ischemia-reperfusion, transplant atherosclerosis, cardiopulmonarybypass, postmenopausal, Kawasaki's disease, Chagas' disease, familyhistory CAD, infections, depression, inactivity, obesity, renal failure,increased CRP, congestive heart failure, and left ventricularhypertrophy.

Embodiment 96: The method according to any one of embodiments 1-95,wherein said measure of endothelial function in said mammal is printedout and/or stored on a tangible medium.

Embodiment 97: The method of embodiment 96, wherein said tangible mediumcomprises a medical record.

Embodiment 98: The method of embodiment 96, wherein said medical recordis a computerized medical record.

Embodiment 99: An apparatus for assessment endothelial function in amammal comprising: a measurement cuff adapted to apply a substantiallypressure to an artery in said mammal; a measurement unit adapted todetect and quantify over one or more cardiac cycles, pressure pulses insaid cuff while said pressure is applied; a controller that is adaptedto apply to the cuff different pressures within a single time period(Tm), and/or to apply substantially constant pressures that differ indifferent time periods (T_(m)) where said controller monitors andadjusts the pressure in said cuff and whose response time is sufficientslow so that the changes in pressure resulting from said cardiac cyclesare not substantially attenuated by said system, and/or that is adaptedto control a pressure source and a valve to provide on-off control ofthe pressure in said cuff; and a processor adapted to determine aplurality of baseline values for a parameter related to endothelialfunction as a function of applied pressure (P_(m)) and to determine aplurality of stimulus-effected values for a parameter related toendothelial function as a function of applied pressure (P_(m)).

Embodiment 100: The apparatus of embodiment 99, wherein said processoris adapted to: determine the area (e.g., a measure proportional to thearea) of the arterial lumen calculated from the baseline pressuremeasurements as a function of transmural pressure P_(tm), wheretransmural pressure is determined as the difference between the baselinemeasured pressures and the external cuff pressure P_(m) and fittingthese data with a first non-linear model to provide a first functiondescribing baseline arterial lumen area as function of transmuralpressure; to determine the area (e.g., a measure proportional to thearea) of the arterial lumen calculated from the stimulus-effectedpressure measurements as a function of transmural pressure P_(tm), wheretransmural pressure is determined as the difference between the measuredstimulus-effected pressures and the external cuff pressure P_(m) andfitting these data with a second non-linear model to provide a secondfunction describing stimulus-effected arterial lumen area as function oftransmural pressure; to use said first function to calculate baselinearterial lumen area (A_(b)) (e.g., a measure proportional to the areaA_(b)) at a transmural pressure substantially equal to the systolicblood pressure; and to use said second function to calculatestimulus-effected arterial lumen area (A_(r)) (e.g., a measureproportional to the area A_(r)) at a transmural pressure substantiallyequal to the systolic blood pressure.

Embodiment 101: The apparatus of embodiment 100, wherein said processoris configured to determine the equivalent ultrasound-based FMD (uFMD)where uFMD is proportional to the square root of the ratio A_(r)/A_(b).

Embodiment 102: The apparatus of embodiment 101, wherein said processoris configured to determine the equivalent ultrasound-based FMD is givenas

uFMD%=[√{square root over ((A _(r) /A _(b))}−1]×100

Embodiment 103: The apparatus according to any one of embodiments99-102, wherein said apparatus is configured to perform the methodaccording to any one of embodiments 1-79, except application of saidstimulus.

Embodiment 104: The apparatus according to any one of embodiments99-102, wherein said apparatus is configured to perform the methodaccording to any one of embodiments 1-79, including application of saidstimulus.

Embodiment 105: The apparatus according to any one of embodiments99-102, wherein said apparatus is configured to perform the methodaccording to any one of embodiments 1-79, including application of saidstimulus where said stimulus comprises restricting flow of blood to alimb by occlusion of a blood vessel.

Embodiment 106: The apparatus of embodiment 105, wherein said apparatusis configured to restrict the flow of blood using a cuff

Embodiment 107: The apparatus of embodiment 106, wherein said apparatusis configured to restrict the flow of blood and apply the pressure onthe artery using separate cuffs.

Embodiment 108: The apparatus of embodiment 106, wherein said apparatusis configured to use the same cuff to occlude the blood vessel and toapply the pressure on the artery.

Embodiment 109: The apparatus according to any one of embodiments 82-83,wherein said apparatus is configured to restrict flow of blood throughthe artery by inflating the restricting cuff to a pressure at least 10mm Hg above measured systolic blood pressure for said mammal.

Embodiment 110: The apparatus according to any one of embodiments105-109, wherein said apparatus is configured to restrict flow of bloodthrough the artery for at least 1 minute.

Embodiment 111: The apparatus according to any one of embodiments99-110, wherein said controller is configured to apply pressure to saidcuff during one or more time periods (T_(m)) by adjusting a pump orother pressure source and/or a proportional release valve to maintain adesired pressure.

Embodiment 112: The apparatus according to any one of embodiments99-111, said controller is configured to stop adjustment of saidpressure during said time period(s).

Embodiment 113: The apparatus according to any one of embodiments99-111, wherein said controller is configured to periodically adjustsaid pressure using an on-off control system during said time period(s).

Embodiment 114: The apparatus according to any one of embodiments99-111, wherein said controller is configured to continuously increaseor decrease said pressure during said time period(s).

Embodiment 115: The apparatus according to any one of embodiments99-111, wherein said controller is configured to maintain pressurewithin a pressure range (AP) around said measurement set point duringsaid time period(s).

Embodiment 116: The apparatus of embodiment 115, wherein said pressurerange (AP) ranges from about 1 mm Hg to about 6 mm Hg, or from about 1mm Hg to about 4 mm Hg, or from about 1 mm Hg to about 3 mm Hg, or fromabout 1 mm Hg to about 2 mm Hg.

Embodiment 117: The apparatus of embodiment 116, wherein said pressurerange (AP) is about 2 mm Hg.

Embodiment 118: The apparatus according to any one of embodiments99-117, wherein apparatus is configured to provide a time intervalduration ranging from about 1 sec, or from about 2 sec, or from about 3sec, or from about 4 sec, or from about 5 sec, or from about 6 sec, orfrom about 7 sec, or from about 8 sec, or from about 9 sec, or fromabout 10 sec, or from about 15 sec up to about 20 sec, or up to about 30sec or up to about 40 sec or up to about 50 sec, or up to about 1 min,or up to about 2 min, or up to about 3 min, or up to about 4 min, or upto about 5 min, or up to about 6 min, or up to about 7 min, or up toabout 8 min, or up to about 9 min, or up to about 10 min, or up to about15 min, or up to about 20 min, or up to about 25 min, or up to about 30min.

Embodiment 119: The apparatus according to any one of embodiments99-118, wherein said controller is configured to monitor and adjust saidpressure at a response time sufficiently slow so that said pressurechanges resulting from said cardiac activity are attenuated by less than10%.

Embodiment 120: The apparatus according to any one of embodiments99-119, wherein said controller is configured to maintain said externalpressure by setting the pressure in said cuff to a value and notaltering external pressure applied to said cuff during the measurementsof pressure variations due to said cardiac activity.

Embodiment 121: The apparatus according to any one of embodiments99-120, wherein said controller is configured to apply an externalpressure equivalent to or below a diastolic pressure determined for saidsubject.

Embodiment 122: The apparatus according to any one of embodiments99-120, wherein said controller is configured to apply an externalpressure below the average diastolic pressure measured for said subjector below an expected diastolic pressure for said subject.

Embodiment 123: The apparatus according to any one of embodiments99-120, wherein said controller is configured to apply an externalpressure below the average diastolic pressure measured for said mammal,but no less than about 10 mmHg below said average diastolic pressure.

Embodiment 124: The apparatus according to any one of embodiments99-123, wherein said controller is configured to apply a constantpressure at different levels during different measurement intervals.

Embodiment 125: The apparatus according to any one of embodiments99-124, wherein the measurement apparatus comprises a hydraulic orpneumatic pump adapted to apply the pressure to said cuff.

Embodiment 126: The apparatus according to any one of embodiments99-125, wherein said response time is reduced by disposing a narrowpressure line between hydraulic or pneumatic pump and said cuff.

Embodiment 127: The apparatus according to any one of embodiments99-126, wherein said apparatus comprises a valve and a pump configuredto provide on-off control of the pressure in said cuff.

Embodiment 128: The apparatus according to any one of embodiments99-127, wherein said apparatus further comprises an accelerometerdisposed to detect movement or vibrations in said cuff or apparatus.

Embodiment 129: The apparatus according to any one of embodiments99-128, wherein said cuff is pressurized with a material selected fromthe group consisting of a gas, a fluid, and a gel.

Embodiment 130: The apparatus according to any one of embodiments99-129, wherein said cuff is adapted to apply pressure substantiallyaround an entire circumference of a limb including the artery.

Embodiment 131: The apparatus according to any one of embodiments99-129, wherein said cuff is adapted to apply a local pressure that doesnot substantially affect other blood vessels in a same limb as theartery.

Embodiment 132: The apparatus according to any one of embodiments99-131, wherein said processor is configured to determine a bloodpressure.

Embodiment 133: The apparatus of embodiment 132, wherein said processoris configured to calculate said external pressure based on one or moreblood pressure measurements and to direct said controller to apply thecalculated external pressure.

Embodiment 134: The apparatus according to any one of embodiments99-133, wherein the controller is configured to induce at least one ofmeasurement round responsive to an indication that a stimulus wasadministered to the artery and at least one of the measurement roundsbefore the indication that the stimulus was administered to the arteryis received.

Embodiment 135: The apparatus according to any one of embodiments99-134, wherein the controller is adapted to apply the pressurecontinuously over at least five cardiac cycles of the patient.

Embodiment 136: The apparatus according to any one of embodiments99-135, wherein the controller is configured to store over the course ofone or more cardiac cycles, changes in pressure in said cuff resultingfrom cardiac activity of said mammal as a function of time.

Embodiment 137: The apparatus according to any one of embodiments99-136, wherein said processor is configured to integrate the value of apressure change over time (calculate the area under a pressure/timecurve) for one or for a plurality of cardiac cycles to determine anintegrated pressure value.

Embodiment 138: The apparatus according to any one of embodiments99-137, wherein said processor is configured to determine the maximum ofthe derivative of the pressure versus time wave form on the rising edgeof a pressure pulse for one or for a plurality of cardiac cycles todetermine a compliance value.

Embodiment 139: The apparatus according to any one of embodiments137-138, wherein said processor is configured to average said integratedpressure value and/or said compliance value over a plurality of cardiaccycles.

Embodiment 140: The apparatus according to any one of embodiments137-138, wherein said processor is configured to determine saidintegrated pressure value and/or said compliance value a single cardiaccycle.

Embodiment 141: The apparatus according to any one of embodiments137-138, wherein said processor is configured to determine saidintegrated pressure value and/or said compliance value and identify amaximum change in said value between a baseline measurement and astimulus-effected measurement.

Embodiment 142: A method of treating a subject for a cardiovascularpathology, said method comprising: assessing endothelial function insaid subject using the method according to any one of embodiments 1-98;and where said method produces a measure indicating impaired endothelialfunction, evaluating said measure in the context of a differentialdiagnosis for a cardiovascular pathology and, where indicated,performing one or more interventions for the treatment of acardiovascular pathology.

Embodiment 143: The method of embodiment 142, wherein said assessingendothelial function is performed using an apparatus according to anyone of embodiments 99-141.

Embodiment 144: The method according to any one of embodiments 142-143,wherein said cardiovascular pathology comprises a pathology selectedfrom the group consisting of atherosclerosis, hypercholesterolemia, highLDL-C, low HDL-C, high lipoprotein (a), small dense LDL-C, oxidizedLDL-C, hypertension, high homocysteine, aging, vasculitis, preeclampsia,metabolic syndrome, variant angina, diabetes, active smoking, passivesmoking, ischemia-reperfusion, transplant atherosclerosis,cardiopulmonary bypass, postmenopausal, Kawasaki's disease, Chagas'disease, family history CAD, infections, depression, inactivity,obesity, renal failure, increased CRP, congestive heart failure, andleft ventricular hypertrophy.

Embodiment 145: The method according to any one of embodiments 142-143,wherein said cardiovascular pathology comprises atherosclerosis.

Embodiment 146: The method according to any one of embodiments 142-145,wherein said intervention is selected from the group consisting ofprescribing and/or administering ACE inhibitors, prescribing and/oradministering angiotensin receptor blockers, prescribing and/oradministering endothelin blockers, prescribing and/or administeringstatins, prescribing and/or administering tetrahydrobiopterin,prescribing and/or administering folates, improving insulin sensitivity,LDL reduction, HDL augmentation, prescribing and/or administeringantioxidants, prescribing and/or administering estrogen, prescribingand/or administering L-arginine, prescribing and/or administeringdesferoxamine, prescribing and/or administering glutathione,homocysteine reduction, lowering CRP, and reducing free fatty acid flux.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic illustration of a system 100 for assessingarterial endothelial function in a mammal.

FIG. 2 shows distension of the brachial artery recorded by M-mode walltracking. Top: Distension waveform under normal conditions. Bottom: Whenthe transmural pressure is decreased by 80 mm Hg using an external cuff,the maximum distention of the artery increases more than twenty-foldover baseline conditions.

FIG. 3 shows typical single pulse waveforms obtained from the pressurecuff when inflated to 70 mm Hg. Both the amplitude and slope of therising edge of the pulse increase markedly after endothelialstimulation. This individual thus exhibits intact endothelial response.Administration of the NO synthase inhibitor L-NAME greatly attenuatesthis response, suggesting that the measurement is primarily sensitive toNO-mediated vasodilation.

FIG. 4 shows results of a study of the effects of a five minute cuffocclusion on the area (upper panel) and maximum derivative of the areavs. time curve (lower panel). Both quantities increase markedly aftercuff release but have returned to baseline levels after 25 minutes.

FIG. 5 shows results of a study of the effects of two serial five minutecuff occlusions on the area (upper panel) and maximum derivative of thearea vs. time curve (lower panel). Both quantities increase markedlyafter cuff release but have returned to baseline levels afterapproximately 10 minutes.

FIG. 6 shows results of a study of the same individual in which the sameprotocol is performed except that the cuff is not inflated tosuprasystolic levels. Some natural drift in the baseline signals isevident, but the magnitude of this variation is far less than theresponse elicited by reactive hyperemia.

FIG. 7 provides a block diagram of a control 200 (111) in accordancewith one illustrative embodiment of the present invention.

FIG. 8 provides a schematic view of one embodiment of apneumatic/hydraulic unit 214 shown in FIG. 7.

FIG. 9 provides flow chart illustrating typical acts performed in ameasurement of the effect of a stimulus on endothelial function.

FIG. 10 shows the fractional change in pulse amplitude (proportional toarea) observed relative to baseline for all studies analyzed. It isclear that the method detects much larger changes in the cases where RHor NG is used as stimulus than when no stimulus is applied. The factthat there is totally unambiguous distinction between thestimulus-present versus NS studies in all cases for the time points inthe range of 8-10 minutes is extremely encouraging.

FIGS. 11A-11C show the results of concurrent measurement of arterydiameter (using ultrasound imaging on right arm) and volume distention(using our method). The curves quantified by the left axes, show thefractional increase in volume distention measured using our method. Thecurves quantified by the right axes are arterial diameter measurementsobtained using ultrasound. The stimulus at t=0 was 400 μg of sublingualnitroglycerin. The diameter measurements exhibit much higher variance,owing to the extreme sensitivity of the method to slight motion of thesubject. As in other studies, these results show that our method is 5times more sensitive to dilation than diameter measurements

FIG. 12 illustrates one embodiment of a system that uses an on-offcontrol system to set the cuff pressure to a constant value duringmeasurement. This is effected by a microcontroller that actuates a pump(or other pressure source) and a valve.

FIG. 13 shows a photograph of a portable prototype device (top) andclose-up (bottom).

FIG. 14 illustrates one embodiment of a system that uses a variablecontrol system to set the cuff pressure to a constant value certainperiods of a measurement or pre-measurement phase. This is effected by amicrocontroller that actuates a pump (or other pressure source) and avalve.

FIG. 15 shows the typical decrease in cuff pressure during themeasurement interval owing to the displacement of tissue under the cuff.The analysis method preferably takes this characteristic into account.

FIG. 16 illustrates the change in arterial compliance with transmuralpressure (blood pressure minus cuff pressure). These data were obtainedusing intraarterial ultrasound and blood pressure measurement.

FIG. 17 illustrates various approaches to improve consistency of meanmeasurement pressure by addressing variations in pressure due tocompression and conformation of the tissue under the cuff. Themeasurement interval is divided into two segments. Each is assigned adifferent servo range, or a different servo mechanism is employed duringeach.

FIG. 18 illustrates components of a home health monitoring systemincorporating methods and/or devices described herein.

FIG. 19, left shows a photograph of an iHealth BP5 wireless bloodpressure cuff. The cuff firmware is modified to allow users to executethe cFMD measurement protocol. Right: Measurement application running onan iPhone 5 that obtains the pressure waveforms from the cuff viaBluetooth.

FIG. 20 shows an illustration of approaches to improve consistency ofmean measurement pressure by addressing variations in pressure due tocompression and conformation of the tissue under the cuff. The signal inthe top panel is acquired with a large servo threshold pressuretolerances of ΔP₁=ΔP₂=10 mmHg with respect to the setpoint of 70 mmHg.Subsidence of tissue under the cuff leads to a drop of over 7 mmHg belowthe set-point over the first 15 s. To yield the data in the bottompanel, the servo threshold is set to ΔP₁=2 mmHg for the first T₁=10 s,and ΔP₂=4 mmHg for the remaining T₂=20 s. These settings lead, in thiscase, to a stabilization of the signal close to the set-point during thefirst 10 s. While the relaxation of the pressure bounds during the last20 s does not have an effect for this time series, it generally reducessignal disruption due to servo action during the later segment of theacquisition period.

FIG. 21 shows a scatter plot of measurements of cFMD % vs. uFMD % forN=27 total subjects. We observe a correlation coefficient of r=0:55,which is statistically significant with p=0:003.

FIG. 22 shows a scatter plot of measurements of cFMD % vs. uFMD % forN=15 total subjects. We observed a correlation coefficient of r=0:82,which is statistically significant with p=0:0002. These subjects are thesubset of those in FIG. 21 that exhibited systolic blood pressures ofless than or equal to 140 mmHg.

FIG. 23 illustrates an arctangent model relating artery cross sectionalarea to transmural pressure. These curves are applicable to anindividual with a blood pressure of 122/77 mmHg.

FIG. 24 shows a scatter plot of measurements of cFMD % vs. uFMD % forN=27 total subjects. We observe a correlation coefficient of r=0.55,which is statistically significant with p=0.003.

FIG. 25 shows a scatter plot of measurements of cFMD % vs. uFMD % forN=15 total subjects. We observe a correlation coefficient of r=0.82,which is statistically significant with p=0.0002. These subjects are thesubset of those in FIG. 24 that exhibited systolic blood pressures ofless than or equal to 140 mmHg.

FIG. 26 Adams et al. observed a marked correlation (r=0.41) betweenendothelium-dependent arterial dilation (EDD) andendothelium-independent dilation (EID) in 800 subjects. When thosesubjects at higher risk of atherosclerosis were removed (diabetics aswell as those with a history of tobacco smoking), the correlationcoefficient fell to 0.24. Our results are consistent with thesefindings, although we do not think the attribution to smooth muscledysfunction is correct. Rather, we think that the method used to measureEDD is confounded by arterial stiffness differences between subjectsthat contribute towards the correlation shown in this plot.

DETAILED DESCRIPTION

In various embodiments, methods and devices are provided fornon-invasively assessing arterial endothelial function in a mammal(e.g., a human or a non-human mammal), particularly in response to astimulus. The change in endothelial function (or lack of change) inresponse to particular stimuli provides a measure of the vascular healthof the subject.

In certain embodiments the method and devices are configured to providecuff-based flow-mediated vasodilation (cFMD) measurements, however, invarious embodiments, methods and apparatus are provided to convert cFMDmeasurements to equivalent ultrasound-based flow-mediated FMD (uFMD)measurements, where ultrasound imaging or vessel wall tracking are usedto determine the diameter of an artery before and after the delivery ofendothelial stimulus.

Determination of cFMD Measurements and Conversion of uFMD Measurements.

Consider FIG. 1 which provides a schematic representation of the crosssection of the human upper arm 112 enclosed in an inflated bloodpressure cuff 101. In conventional blood pressure measurement, the cuffis initially inflated above the systolic blood pressure. This appliespressure to the skin surface 113 which compresses the arm and thecontents thereof (e.g., humerus 114, brachial artery 115, etc.) causingthe underlying arteries 115 to collapse. The pressure in the cuff inthis case is purely determined by the external pressure applied by theair in the cuff.

Consider the case where the cuff is inflated to a pressure belowdiastolic pressure. This distorts the shape of the artery causing theartery to partially collapse. As the pressure in the artery increasesduring the course of the natural blood pressure pulse (i.e., exceeds thediastolic pressure), the flattened artery expands. As a consequence ofthe near incompressibility of human tissue and body fluids, the pressurein the cuff increases in proportion to the increase in arterial crosssectional area. By measuring the pressure in the cuff, it is thuspossible to obtain a measure of arterial caliber.

Consider an illustrative example, where 70 mm Hg pressure is applied tothe cuff when the subject's diastolic pressure is 80 mm Hg. In certainembodiments this is accomplished by attaching a constant pressure source103 to the cuff that provides the 70 mm Hg pressure. In variousembodiments the constant pressure source 103 utilizes a hydraulic orpneumatic pump or pressurized gas, or a fluid reservoir. Such sourcestypically utilize a servo/valve mechanism to maintain the pressure setpoint, and this servo can be under control of a pressure controller 105.In some embodiments, a pump and valve are actuated by a control systemin order to keep the pressure within an acceptable range (e.g. ±5 mm Hg)about the set point.

To preserve pressure signals resulting from cardiac activity (i.e.,cardiac cycle(s)) it is desirable that the pressure source notsubstantially cancel out the changes in cuff pressure due to theincrease in area of the flattened vessel. This may be achieved byincreasing the time constant of the system response of theservo/pressure controller system and/or more simply by placing a flowresistor 116 between the pressure source and the cuff. In the simplestimplementation, a long thin tube (e.g., 1 m (or other) length of thinintervening tubing that serves as a pneumatic low pass filter) canprovide this resistance. Another option is to decouple the constantpressure source from the cuff once the cuff has reached its targetpressure.

In various illustrative embodiments, the time constant of the pressuresystem is sufficiently slow relative to pressure changes introduced bythe cardiac cycle that pressure changes resulting from cardiac activity(e.g., pulse-associated pressure changes) are attenuated by less than20%, or less than about 15%, or less than about 10%, or less than about5%, or less than about 1% of the maximum pressure change. Similarly asubstantially constant pressure is a pressure that when averaged over asufficiently long time period that pulse-induced pressure changes areaveraged out, the average pressure applied to the cuff over the desiredtime period various by less than 20%, more preferably less than about15%, or less than about 10%, most preferably less than about 5%, 3%, 2%,or 1% of the applied pressure.

In various embodiments the pressure in the cuff is measured using apressure transducer (pressure sensor) 102. One illustrative suitablepressure sensor is the Millar catheter pressure sensor (Mikro-tip,Millar Instruments, Houston, Tex.). The output signal of transducer canbe amplified (e.g., using an instrumentation amplifier such as AD627,Analog Devices, Inc., Norwood Mass.), optionally low-pass filtered(e.g., using 8th Order elliptic Filter, LTC-1069-6, Linear TechnologyCorp., Milpitas, Calif.), and then digitized (e.g., at 1 kHz using a A/Dconverter PCI card (NI-6035, National Instruments, Austin, Tex.).

The digitized signal can be directly interpreted as a quantityproportional to the area of the arterial lumen as long as the pressurein the cuff is less than the systolic pressure of the subject, and aslong as the pressure at the outlet of the pressure source is heldsubstantially constant. The pressure source we used in one prototype(Hokanson E20, Bellevue, Wash.) provides servo regulation that is toofast to allow its direct application to the cuff without attenuating thesignal due to the expansion of the arterial lumen. Consequently, weemployed a 1 m length of thin intervening tubing to serves as apneumatic low pass filter.

An illustrative, but non-limiting, protocol can involve the followingsteps (see also flow chart in FIG. 9):

1. The subject is seated or lies supine and rests briefly, e.g., forfive minutes.

2. The subject's blood pressure is measured.

3. The cuff is inflated to at or, somewhat below, the diastolic pressure(e.g., 10 mm Hg below the diastolic blood pressure) and the pressuresignal is recorded to determine a baseline value for a parameter relatedto endothelial function in said mammal (e.g. integrated pressure as afunction of time).

4. A stimulus is applied to the subject.

5. A pressure signal is recorded with the cuff inflated to at or,preferably somewhat below, the diastolic pressure (e.g., 10 mm Hg belowthe diastolic blood pressure) and the pressure signal is recorded todetermine a stimulus-effected value for a parameter related toendothelial function in said mammal (e.g. integrated pressure as afunction of time).

6. The stimulus-effected value of the parameter is compared to thebaseline value of the parameter to determine presence, absence, and/ordegree of endothelial response to said stimulus.

Any of a number of different types of stimuli can be used. Typically,however, the stimulus is one expected to have an effect on endothelialfunction in a mammal. Such stimuli include, but are not limited toocclusion of blood flow, application of drugs (e.g., NO agonists,β₂-adrenergic agonists such as albuterol, acoustic/mechanical tissuevibration, ultrasound stimulus, and the like).

One illustrative non-limiting protocol where the stimulus comprisesocclusion of blood flow can involve the following steps:

1. Subject is seated or lies supine and rests, e.g., for five minutes.

2. The subject's blood pressure is measured.

3. The cuff is inflated to 10 mm Hg below the diastolic blood pressurefor one minute. During this time, the pressure signal is recorded todetermine a baseline value for a parameter related to endothelialfunction in said mammal.

4. The cuff is deflated for 30 seconds to allow blood flow to returntoward normal.

5. The cuff is inflated to, e.g., 40 mm Hg above systolic pressure forfive minutes.

6. The cuff is released to allow reactive hyperemia to ensue, e.g., arelease for 35 seconds.

7. The cuff is inflated to, e.g., 10 mm Hg below the diastolic bloodpressure for three minutes. During this time, the pressure signal isrecorded to determine a stimulus-effected value for a parameter relatedto endothelial function in said mammal.

8. The stimulus-effected value of the parameter is compared to thebaseline value of the parameter to determine presence, absence, and/ordegree of endothelial response to said stimulus.

A second illustrative non-limiting protocol where the stimulus comprisesocclusion of blood flow can involve the following steps:

1. Subject is seated or lies supine and rests for five minutes.

2. The cuff begins inflating rapidly to, e.g., 20 mmHg.

3. The cuff is inflated at a rate of 3-6 mmHg/s and the pulse waveformis monitored and recorded throughout.

4. The maximum amplitude of the pulse waveform is detected as the cuffpressure becomes close to mean arterial pressure (MAP).

5. Cuff inflation continues until the amplitude of the pulse waveform isattenuated below a certain threshold (usually a fraction of the maximumamplitude obtained near MAP).

6. The cuff is deflated suddenly. Alternatively, the cuff is deflatedslowly, e.g., at a rate of 3-6mmHg/s.

7. After a rest period of 30 seconds, the blood flow in the limb isoccluded, e.g., for 60-300 s.

8. The occlusion is released. A rest period of, e.g., 45 secondsfollows.

9. Steps 2-6 are repeated to obtain a post stimulus measurement.

10. The stimulus-effected value of the parameter is compared to thebaseline value of the parameter to determine presence, absence, and/ordegree of endothelial response to said stimulus. The analysis of thewaveforms recorded yields systolic blood pressure (SBP), diastolic bloodpressure (DBP), an estimate of MAP (the maximum amplitude of theoscillometric waveform). Comparison of the waveforms to estimateendothelial function are referenced to the values of SBP, DBP and MAPobtained.

Calculation of uFMD from cFMD.

In various embodiments the methods described above can be performed in amanner that facilitates conversion of cFMD measurements to equivalentultrasound-based flow-mediated FMD (uFMD) measurements, e.g., uFMDmeasurement where ultrasound imaging or vessel wall tracking are used todetermine the diameter of an artery before and after the delivery ofendothelial stimulus. Thus, methods and devices are provided that permitmeasurement and conversion of cFMD measurement to equivalent uFMDmeasurements, e.g., as described in Example 3.

Accordingly in various embodiments the methods described above cancomprise:

a) applying external pressure P_(m) to an artery for a period of timeT_(m), and determining, over the course of one or more cardiac cycles inperiod T_(m), changes in pressure in a cuff resulting from cardiacactivity of the mammal, or an artificially induced arterial pulse, todetermine values for a baseline parameter related to endothelialfunction in the mammal, where said baseline parameter is determined forat least three different external pressure points (P_(m)) (or at least 3different external pressure points during a continuous pressurevariation) by varying the external pressure P_(m) during period of timeT_(m), and/or by determining said baseline parameter over a plurality oftime periods T_(m) having different external pressure points (P_(m)), toprovide a plurality of baseline values for said parameter related toendothelial function as a function of applied pressure (P_(m));

b) applying a stimulus to the mammal; and

c) applying external pressure P_(m) to an said artery for a period oftime T_(m), and determining over the course of one or more cardiaccycles in period T_(m), changes in pressure in the cuff resulting fromcardiac activity of the mammal, or an artificially induced arterialpulse, to determine values for a stimulus-effected parameter related toendothelial function in said mammal, where said stimulus-effectedparameter is determined for at least three different external pressurepoints (P_(m)) (or at least 3 different external pressure points duringa continuous pressure variation) by varying the external pressure P_(m)during period of time T_(m), and/or by determining said parameter over aplurality of time periods T_(m) having different external pressurepoints (P_(m)), to provide a plurality of stimulus-effected values forthe parameter related to endothelial function as a function of appliedpressure (P_(m)); wherein the baseline values are determined frommeasurements made when said mammal is not substantially effected by thestimulus and differences in said baseline values and saidstimulus-effected values provide a measure of endothelial function insaid mammal.

It will be recognized that in certain embodiments the variations inpressure (e.g., varying external pressures) can be a multiple discretepressures permitting measurements to be made at those multiple discretepressures. In certain other embodiments the variations in pressure canbe continuous variations in pressure, and the measurements are madeduring the continuously varying pressure. Where the pressure is varied(e.g., continuously varied) the variation can be monotonic ornon-monotonic.

In certain embodiments the methods described above (and in the examples)can comprise:

d) determining the area of the arterial lumen calculated from thebaseline pressure measurements as a function of transmural pressureP_(tm), where transmural pressure is determined as the differencebetween the baseline measured pressures and the external cuff pressureP_(m) and fitting these data with a first non-linear model to provide afirst function describing baseline arterial lumen area as function oftransmural pressure;

e) determining the area of the arterial lumen calculated from thestimulus-effected pressure measurements as a function of transmuralpressure P_(tm), where transmural pressure is determined as thedifference between the measured stimulus-effected pressures and theexternal cuff pressure P_(m) and fitting these data with a secondnon-linear model to provide a second function describingstimulus-effected arterial lumen area as function of transmuralpressure;

f) using said the function to calculate baseline arterial lumen area(A_(b)) at a transmural pressure substantially equal to the systolicblood pressure;

g) using said second function to calculate stimulus-effected arteriallumen area (A_(r)) at a transmural pressure substantially equal to thesystolic blood pressure; and

h) determining the equivalent ultrasound-based FMD (uFMD) measure whereuFMD is proportional to the square root of the ratio A_(r)/A_(b).

In certain embodiments the cuff is fully calibrated so that luminal areacan be calculated. However, in various embodiments, this is not requiredsince the ratio of uncalibrated area estimates is independent of thiscalibration. Thus, in various embodiments, the term “area” refers to aquantity proportional area even if it is not an absolutely calibratedarea.

In certain embodiments, the equivalent ultrasound-based FMD is given as:

uFMD%=[√{square root over ((A _(r) /A _(b))}−1]×100

In various embodiments the first non-linear model and/or the secondnon-linear model are the same type of function. In certain embodimentsthe first non-linear model and/or the second non-linear model areselected from the group consisting of a 2-parameter model, a 3 parametermodel, a four parameter model, a 5 parameter model, and a six parametermodel. In certain embodiments the first non-linear model and/or thesecond non-linear model are three parameter models. In variousembodiments the first non-linear model and/or the second non-linearmodel are arctangent models.

In various embodiments step (a) and/or step (b) of the method comprisesdetermining values for a baseline parameter and/or values for astimulus-effected parameter for at least 3 different pressure points(P_(m)), or for at least 4 different pressure points (P_(m)), or for atleast 5 different pressure points (P_(m)), or for at least 6 differentpressure points (P_(m)), or for at least 7 different pressure points(P_(m)), or for at least 8 different pressure points (P_(m)), or for atleast 9 different pressure points (P_(m)), or for at least 10 differentpressure points (P_(m)).

In various embodiments step (a) and/or step (b) of the method comprisesdetermining values for a baseline parameter and/or values for astimulus-effected parameter for at least 3 different pressure points(P_(m)) during a continuous pressure variation, or for at least 4different pressure points (P_(m)) during a continuous pressurevariation, or for at least 5 different pressure points (P_(m)) during acontinuous pressure variation, or for at least 6 different pressurepoints (P_(m)) during a continuous pressure variation, or for at least 7different pressure pointss (P_(m)) during a continuous pressurevariation, or for at least 8 different pressure points (P_(m)) during acontinuous pressure variation, or for at least 9 different pressurepoints (P_(m)) during a continuous pressure variation, or for at least10 different pressure points (P_(m)) during a continuous pressurevariation.

In various embodiments the external pressure is less than the meanarterial pressure. In certain embodiments external pressure is less thanthe mean diastolic pressure.

In certain embodiments the external pressure (Pm) ranges from about 20mmHg up to about 80 mmHg.

In certain embodiments the external pressure points (P_(m)) used todetermine the baseline values are substantially the same as the externalpressure points (PM) used to determine the stimulus-effected values orthe external pressure points (P_(m)) used to determine the baselinevalues are different than the external pressure points (PM) used todetermine the stimulus-effected values.

In various embodiments the baseline parameter is determined for at leastthree different external pressure points (P_(m)) by varying the externalpressure P_(m) during period of time T_(m), and/or by determining saidbaseline parameter over a plurality of time periods T_(m) havingdifferent external pressure points (P_(m)).

As noted above, in various embodiments, the pulse waveform is determinedthroughout the cuff inflation and/or deflation. In certain embodimentsthe cuff inflation and/or deflation is at a rate of about 3-6 mm Hg persecond. Analysis of the pulse waveform yields systolic blood pressure(SBP), diastolic blood pressure (DBP), an estimate of MAP (the maximumamplitude of the oscillometric waveform). Comparisons of the waveformsto estimate endothelial function are referenced to the values of SBP,DBP and MAP obtained. In certain embodiments the pulse waveformsobtained in this way can be compared between baseline and post-stimulusconditions, either by direct comparison of pulse characteristics atcorresponding pressure levels, or by first fitting models (e.g. anon-linear model such as a 2 parameter model, a 3 parameter model, afour parameter model, a 5 parameter model, and a six parameter model,etc.) to each set and comparing the parameters obtained from the fittedmodels those models. Illustrative pulse characteristics (parameters)related to endothelial function include, but are not limited to the peakvalue of a pressure pulse or the maximum peak value of a number ofpressure pulses, or the average or median peak value of a number ofpressure pulses. Other illustrative parameters include, but are notlimited to the area under a pulse in a pressure versus time plot (i.e.,the integrated value of pressure as a function of time) for a pulse, thepeak integrated value of a series of pulses, or the average or medianintegrated value of a series of pulses. Another useful parameter is thederivative of the area vs. time waveform, e.g., the maximum of thisderivative on the rising edge of the pulse. If the endothelial stimulusdoes not affect systemic systolic or diastolic blood pressure (which isa very reasonable assumption), we can assume that the pressure at thepoint at which the slope of the area versus time curve is maximal isapproximately the same before and after endothelial stimulus. In thiscase, this slope is an approximately proportional to dA/dP, which is thecompliance of the vessel (A and P represent area and pressure,respectively). Compliance is the fundamental quantity reduced by thesmooth muscle relaxation that is a consequence of healthy endothelialresponse. It constitutes a extremely valuable “root cause” metric.

In various embodiments blood pressure (BP) and cFMD can be measured inone set of readings. The advantage of this approach is that the featuresrelated to BP are provided in the same continuous measurement as thefeatures used for cFMD determination. If BP varies between baseline andpost-stimulus intervals, this can be detected and the measurement can bedisqualified or compensated for, since the baseline and post-stimulusdata sets can be compared as a function of transmural pressure, notmeasurement pressure.

Accordingly, in one illustrative, but non-limiting approach, the cuff isinflated (perhaps slowly, e.g., at 3-6 mm Hg/s) to a pressure in excessof the peak of the oscillometric waveform. When inflation is to apressure in excess of the systolic pressure, this pressure may beidentified as a certain level of diminution of the amplitude of thewaveform, as in the standard oscillometric methods. Typically inflationwill be to a pressure that identifies the peak of the oscillometricwaveform, which is generally taken to represent the mean arterialpressure (MAP). After identifying the systolic BP and MAP, the diastolicBP may be calculated according to standard methods used to implementoscillometric determination of BP (e.g., as reviewed and discussed byBabbs (2012) BioMedical Engineering OnLine, 11:56 DOI:10.1186/1475-925X-11-56i.). Additionally, or alternatively, in certainembodiments, the diastolic blood pressure (DBP) can be identified byanalyzing Korotkoff sounds obtained using an audio sensor (stethoscope,phonocadiogram), or ultrasound probe. In certain embodiments afterreaching the inflation pressure, the presser can be slowly released andone or more sets of measurements can be made to calculate cFMD and/orMAP, and/or DBP during cuff deflation. Alternatively, the cuff cansimply be deflated, having obtained all measurements determined duringslow inflation.

In certain embodiments for cFMD determination, a comparison of pulsewaveform characteristics between baseline and post-stimulus intervals at“corresponding pressures” can be a comparison made where thecorresponding pressures are transmural pressures calculated using the BPvalues obtained as described above, rather than comparisons based oncorresponding measurement (cuff) pressures. In certain embodiments, whentwo fitted models are compared, the comparisons can be based ontransmural pressure as the pressure variable.

In certain embodiments measurements of the same mammal are obtainedseveral times. Then previous measurements can be used to establish thetone state of the vessel at the current time at baseline and the degreeof current dilation (tone state) at baseline can be used to adjust thecFMD and BP measurements to the current vessel tone state, in a mannersuggested by Babbs (2012), supra. In certain embodiments the maximaldilated state is measured by stimulating the vessel with an NO donorsuch as nitroglycerin and/or the constricted state is measured bystimulating the vessel with a vasoconstrictive substance such asphenylephrine. In certain embodiments the dilatory and constrictorypotential of the vessel and tone state are inferred by measurementsobtained with the cuff elevated at different vertical levels withrespect to the heart. This varies the hydrostatic pressure on the walland enables characterization of vessel mechanics.

In view of the foregoing, it will be recognized that cFMDdeterminations, using a comparison of pulse waveform characteristicsbetween baseline and post-stimulus intervals at corresponding transmuralpressures calculated using the determined BP values can be used tocalibrate the threshold points used for oscillometric determination ofthe DBP, as may be understood from Babbs (2012), supra.

The oscillometric method has been relatively well validated as a measureof mean arterial pressure, which is indicated by the peak of theoscillation amplitude envelope. Use of the oscillometric method inscreening for high blood pressure has proven problematic, because theaccuracy of systolic and diastolic end points has been questioned anddoubted. However, in certain embodiments, a model describing thedependence of the oscillometric characteristic on vessel tone can beused to refine thresholds for oscillometric determination of DBP. Oneillustrative such model is described by Babbs (2012) BioMedicalEngineering OnLine, 11:56 DOI: 10.1186/1475-925X-11-56i. Determinationsof vessel tone using the methdos and devices described herein can beused to change the calibration of the thresholds used for DBPdetermination by the oscillometric method and provide accurate systolicand diastolic end points. In cases where maximally-dilating stimuli suchas nitroglycerin (NG) are used, subsequent studies without NG may usethe NG study as reference of maximum dilation. The endothelium-mediateddilation measurements calibrated for the maximum attainable dilation. Incases where NG is employed at a similar time to the endothelial functionstudy, the baseline tone, and tone after endothelial stimulus, may beassessed by comparison to the maximally dilated state.

As noted above, amount of FMD measured by any method is dependent on theinitial vessel tone (tension state of the arterial vascular smoothmuscle). A fully dilated vessel cannot, by definition, exhibitadditional dilation in response to a stimulus. Ideally, a stimulus suchas nitroglycerin can be applied to assess the maximum dilation capacityof the vessel. However, when multiple measurements are performed on anindividual over time, the natural variation of these states can be“learned” by an algorithm. This may be used to obtain refined estimatesof not only endothelial function, but also DBP and MAP, which are alsosubstantially dependent on arterial tone, and, to a lesser extent, heartrate. By repeatedly applying protocols described above, a large portionof the characteristic curve: (luminal area change as a function of pulsepressure, as represented by the distension waveform recorded as pressurechanges in the cuff) versus transmural pressure, is acquired underseveral different natural conditions. This enables an algorithm to learnthis characteristic, which as Bank et al. (1999) Circulation, 100:41-47, have proven, is dependent on vessel tone. Specifically, the dataobtained from the baseline recordings can improve measurements of SBP,DBP and MAP, regardless of the intactness of endothelial function in themammal. The major effect of prevailing tone at the time of BPmeasurement, on values of DBP obtained via the oscillometric method, hasbeen demonstrated by Babbs, and others. It is also known that theseestimates are also dependent on pulse shape and heart rate. All of thisinformation can be recorded in the baseline and post-stimulus pressurewaveforms acquired by various embodiments of the invention describedherein, and enables an algorithm to more accurately estimate both BP andendothelial function based on estimating the tone state of the vessel.

A fundamental advantage of the present methods over traditional measuresof flow-mediated vasodilation (FMD) is the increased sensitivity thatcomes from measuring parameters related to arterial cross-sectional arearather than radius, since area is approximately proportional to thesquare of the radius. Also, by decreasing the transmural pressure on theartery using an external cuff inflated just below diastolic levels, thedistensibility of the artery is increased by more than an order ofmagnitude (Bank et al. (1995) Circ. Res., 77(5): 1008-1016; Bank et al.(1999) Circulation, 100: 41-47; Kim et al. (2004) Ultrasound in Medicine& Biology, 30: 761-771). As FIG. 2 illustrates, we have observed thiseffect in our laboratory using M-mode ultrasound to track the arterialwall. These two factors combined lead impart exceptionally highsensitivity to the methods and devices described herein.

In various embodiments, the R-wave of the patient ECG can be used as atiming reference to facilitate the analysis of individual pulses. Incertain embodiments it is possible, however, to perform such analysisusing the pressure waveform alone.

FIG. 3 shows typical single pulse waveforms obtained by measuringpressure changes in the cuff In an artery with intact endothelialfunction, both pulse height (maximal cross-sectional arterial area) andcompliance (maximum slope of the rising edge) increase markedly overbaseline. When NO synthase is blocked via the inhibitor L-NAME, bothpulse height and slope increases are greatly attenuated. FIG. 4illustrates how a five minute cuff occlusion and the ensuring reactivehyperemia lead to major increases in area change per pulse and themaximum derivative of the area per pulse. Both metrics return tobaseline levels after 20 minutes. FIG. 5 confirms the repeatability ofthe protocol by illustrating the effects of a series of two cuffocclusion periods. We see from FIG. 6 that only a small slow drift inthe measured quantities occurs when no reactive hyperemic stimulus isapplied.

Two Cuff Method

In certain embodiments, it may be preferred to obtain endothelialfunction measurements in segments of arteries that are not subject toischemia during cuff occlusion. To allow such studies to be performed,two cuffs, or a single cuff that is segmented into two bladders may beused. The proximal cuff is used for measurement and is inflated to apressure that does not fully occlude the vessel. The distal bladder isinflated to suprasystolic pressure during the stimulus interval in orderto create downstream ischemia.

In another embodiment, two cuffs are used on the same limb and inflatedto some substantially constant pressure. Pressure pulses resulting fromcardiac activity (cardiac cycles) are detected in each cuff. The metricof vasorelaxation used is the transit time of the pulse between the twocuffs. When the vessel is dilated, the transit time decreases. Again thetransit time measurement can be initially made to establish a baselinevalue. The subject can be administered a stimulus, and the transit timedetermined again to determine a stimulus-effected transit time.

An illustrative, but non-limiting, protocol can involve the followingsteps:

1. The subject is seated or lies supine and rests briefly, e.g., forfive minutes.

2. The subject's blood pressure is measured.

3. Both cuffs are inflated to at or, preferably somewhat below, thediastolic pressure (e.g., 10 mm Hg below the diastolic blood pressure)and the pressure signal in each cuff is record to calculate a baselinetransit time for a pressure pulse from the medial cuff to the distalcuff.

4. A stimulus is applied to the subject.

5. A pressure signal is recorded with both cuffs inflated to at or,preferably somewhat below, the diastolic pressure (e.g., 10 mm Hg belowthe diastolic blood pressure) and the pressure signal in each cuff isrecord a stimulus-effected transit time for a pressure pulse from themedial cuff to the distal cuff.

6. The stimulus-effected value of the transit time is compared to thebaseline value of the transit time to determine presence, absence,and/or degree of endothelial response to said stimulus.

In various embodiments the systems and methods described herein aresuitable for ambulatory use. Inflation of the cuff, for example, can beperformed using a battery powered pump, or using replaceable/refillablegas cartridges. The subject can be alerted before a scheduledmeasurement commences and instructed to remain still and sit or liedown.

The foregoing protocols are intended to be illustrative and notlimiting. For example, while the foregoing methods are described withrespect to measurement of pressure pulses in the cuff resulting fromcardiac activity in the subject, they need not be sol limited. Thus, incertain embodiments, the methods involve recording artificially inducedarterial pressure pulses. Methods of artificially inducing arterialpressure pulses are known to those of skill in the art. For example,Maltz and Budinger (2005) Physiol. Meas. 26: 293-307 describe the use alinear actuator to induce an artificial arterial pressure pulse (seealso U.S. Pat. No: 8,666,472). The actuator described herein employed alinear motor (from Baldor Electric Co., Fort Smith, Ark.), the actuatingstem of which was adapted to make contact with the skin to introduce anartificial pulse. An applanation tonometer (SPT301, Millar Instruments,Inc., Houston, Tex.) at the free end of the stem sensed the appliedforce and allowed for closed-loop control of the force waveform.

In another embodiment, a cuff attached to a high bandwidthelectropneumatic converter can be used to induce an artificial arterialpressure pulse. One illustrative electropneumatic converter is describedby Tanaka et al. (2003) Engineering in Medicine and Biology Society,Proceedings of the 25th Annual International Conference of the IEEE, 4:3149-3152. Tanaka et al. a disk-type cuff for local pressurization and anozzle-flapper type electro-pneumatic converter (EPC) for thecuff-pressure control.

These embodiments are illustrative and not limiting. In view of theteachings provided herein, numerous methods to induce an artificialarterial pressure pulse are available to one of skill in the art. Incertain embodiments even a standard cuff can be sufficient to induce asuitable pressure disturbance.

The systems described herein can be applied to arteries in the upperarms (or forelegs), forearms, the wrist, the thighs (hind legs), calves,ankles, and possibly even the neck (carotid arteries). In certainembodiments during the protocol, a second cuff may be applied to thecontralateral limb (to which no endothelial stimulus is applied, or towhich some other stimulus is applied) to serve as reference or to obtaindifferential measurements that elucidate the relative contributions ofvarious vascular response mechanisms mediated by different biochemicalpathways.

In various embodiments the system can be used to evaluate the effects ofother stimuli including, but not limited to the influence of smoothmuscle relaxation agents such as nitroglycerin, the influence of mentalor physical stress, low intensity ultrasound β₂-adrenergic agonists suchas albuterol, acoustic/mechanical tissue vibration, and the like. Invarious embodiments the cuff pressure may be set at different levels(during the measurement phase) to achieve different degrees ofmechanical unloading. This can help to reduce the number of assumptionsrequired for the interpretation of dA/dt as a measure of dA/dP. Aramping of the cuff pressure can also help to characterize the vesselmore thoroughly. In various embodiments to improve signal quality, thecuff may be filled with a liquid or a gel rather than a gas.

In one particular illustrative application, the device, systems, andmethods described herein are well suited for evaluation of subjectsdiagnosed with or at risk for sickle cell disease. In this context it isnoted that the methods are highly suited to children relative toultrasound as they are not very motion sensitive and young children areoften difficult subjects. There is severe disruption of endothelialresponse in sickle cell disease and monitoring this can aid diseasemanagement.

FIG. 1 which provides a schematic illustration of a system 100 forassessing endothelial function in accordance with an illustrativeembodiment of the methods and devices described herein. The systemcomprises a measurement cuff (e.g., blood pressure cuff) 112 that isconfigured for attachment to (around) a limb of a mammal (e.g., an arm,wrist, a leg, an ankle, etc.). The cuff can be fastened by anyconvenient method including, but not limited to a strap, a clip, aVelcro closure and the like. The cuff is used to administer asubstantially constant pressure to the limb.

One or more bladders comprising the cuff are connected to a constantpressure source 103 that applies the constant pressure to the cuff. Thepressure in the cuff in this case is purely determined by the externalpressure applied by the air in the cuff. The pressure source can becoupled to a pressure controller 105 that regulates a valve or otheractuator on the pressure source to regulate the substantially constantpressure applied to the cuff.

A pressure transducer (pressure sensor) 102 is disposed to monitor thepressure in the cuff. The output signal of the pressure sensor is readby a control unit 111 that comprises the circuitry necessary to readand, if necessary, to drive, the pressure sensor. In one illustrativeembodiment, the control unit 111 comprises an amplifier 107 (e.g.,instrumentation amplifier AD627, Analog Devices, Inc., Norwood Mass.)that amplifies the output signal of the pressure transducer, an optionallow pass filter 108 (e.g., 8th Order elliptic Filter, LTC-1069-6, LinearTechnology Corp., Milpitas, Calif.) and a digitizer 109 (e.g., an A/Dconverter PCI card (NI-6035, National Instruments, Austin, Tex.).Another tested embodiment employed a 0.6×0.6 in² MEMS pressure sensor(NPC-1210, GE Novasensor, Fremont, Calif.). The control unit 111 isconfigured to read the pressure from the pressure transducer.

In various embodiments the control unit 111 can be coupled to thepressure controller (e.g., via a signal cable) and thereby regulate thepressure applied to the cuff. As indicated by the dashed lines, invarious embodiments, the controller 111 and pressure controller 105 canbe integrated into a single control unit that both regulates theconstant pressure source and reads the pressure fluctuations resultingfrom cardiac activity. In other embodiments, the controller 111 andpressure controller 105 can be separate units that communication (e.g.,via a signal cable) or that, in certain embodiments, are independentlycontrolled.

In certain embodiments the controller 111 as illustrated in FIG. 1,further comprises a microprocessor 110 (e.g., for signal processingand/or operating the pressure controller). The microprocessor 110however need not be integrated into the controller, but may be a“separate” computer e.g., as described below. In certain embodiments thecontroller comprises a microprocessor that is itself connected to anexternal processor/computer. Thus, in some embodiments, the control unitmay be connected to a computer via a cable for configuration and/or datadownload and/or for communication with an external computer, and/or foroperation of the system.

FIG. 7 provides a block diagram of a control 200 in accordance with oneillustrative embodiment of the present invention. A microprocessor 206optionally serves a central control and integration function controllingthe various units/components therein. As illustrated in FIG. 7, thecontrol unit includes, or is coupled to a pneumatic or hydraulic unit214 (e.g., a unit comprising a pressure source 103 and/or a pressurecontroller 105) that operates to establish a substantially constantpressure in a cuff (cuff 1) via a hydraulic or pneumatic line 218. Incertain embodiments, particularly where a pressure pulse transit time isto be determined, the control unit optionally includes, or is optionallycoupled to a second pneumatic or hydraulic unit 216 (e.g., a unitcomprising a pressure source 103 and/or a pressure controller 105) thatoperates to establish a substantially constant pressure in a second cuff(cuff 2) via a hydraulic or pneumatic line 218. It will be appreciatedthat the pneumatic or hydraulic control units can be used generally toinflate and/or deflate the cuffs as well.

Sensor electronics 222 are provided to send commands to sensortransducer and/or to read a signal from the pressure transducermonitoring pressure in the first cuff (cuff 1). Thus, in certainembodiments, a signal from a first pressure transducer in cuff 1 istransmitted along line 234 to sensor electronics 222, comprising forexample, an amplifier 224, and/or a filter or signal conditioner 226and/or any other electronics useful to drive, read, or transform thepressure transducer signal. An analogue to digital converter (A/D) 202optionally converts the readings of the pressure transducer from cuff 1and/or sensor electronics 222 into digital samples provided tomicroprocessor 206.

Where a second cuff is to be monitored, the control unit optionallyfurther comprises sensor electronics 230 to send commands to sensortransducer and/or to read a signal from the pressure transducermonitoring pressure in a second cuff (cuff 2). Thus, in certainembodiments, a signal from a second pressure transducer in cuff 2 istransmitted along line 236 to sensor 1 electronics 232, comprising forexample, an amplifier 228, and/or a filter or signal conditioner 230and/or any other electronics useful to drive, read, or transform thepressure transducer signal. An analogue to digital converter (A/D) 202optionally converts the readings of the pressure transducer from cuff 2and/or sensor electronics 2232 into digital samples provided tomicroprocessor 206.

In illustrative embodiments, the pressure transducers comprise a sensorsuch as the Millar catheter pressure sensor (Mikro-tip, MillarInstruments, Houston, Tex.) or MEMS pressure sensor such as the NPC-1210(GE Novasensor, Fremont, Calif.), but most low cost sensors used inautomatic sphygmomanometers constitute suitable transducers.

Microprocessor 206 optionally also communicates with display 210, userinput interface 204, and dynamic memory or static memory storage media212 (e.g., disk drive, flash memory, optical memory, etc.). In someembodiments one or more communications lines 208 are used to communicatewith an external computer or any other external unit. Power can beprovided to the unit by an internal or external power supply thatreceives external power through a cable and/or through batteries.

In certain embodiments, the control unit 111/200 can be connected to acomputer via Bluetooth, via a cable, and the like for configuration,control, and/or data download. In certain embodiments, the computer isintegrated into the control unit and microprocessor 206 can function asthe central processing unit of the computer, or another microprocessoris optionally present for such function. The computer can, for example,be dedicated for use with system 200, a personal computer in aphysician's clinic, part of a hospital network and/or a remote computerconnected, for example, through the internet, an intranet, or via a cellphone link. In certain embodiments, for example, a computer networkconnection can be used for may be used for receiving patient data and/orproviding test results to remote locations. In some embodiments thecomputer manages a database of test results classified according todemographic and/or epidemiologic data for the purpose of determiningendothelial dysfunction trends and/or for comparing current test resultsto previously acquired results from same or different patients. In someembodiments, the computer connects with a patient medical record systemsuch as is maintained by a hospital, physician's office, HMO, PPO, andthe like.

FIG. 8 provides a schematic view of one embodiment of apneumatic/hydraulic unit 214 shown in FIG. 7. Pneumatic unit 214includes a pressure source 103 configured to provide output pressure upto a pressure that completely occludes blood flow through a limb orportion of a limb (or other region of a body). Typically pressures canbe delivered that range up to about 200 mm Hg, up to about 250 mm Hg, upto about 300 mm Hg, up to about 350 mm Hg, up to about 380 mm Hg, or upto about 400 mm Hg or greater. Valve 302 optionally controls flow of apressurized gas (e.g., air or other pressurized gas or gas mixture), ora pressurized fluid or gel from pressure source 103 to cuff 100. A valve302 is optionally shut off after a desired substantially constantpressure is applied to the cuff. Another valve 304 is optionallyprovided to vent the cuff through outlet port/waste line 306 to reducepressure or deflate the cuff.

An optional valve 308 can be provided to restrict flow to the cuff andthereby slow the response time of the pneumatic/hydraulic unit so thatpressure regulation does not substantially attenuate pulses produced inthe cuff by cardiac activity. A pressure line 106 carries the gas,fluid, or gel to the cuff whereby the cuff is inflated or deflated. Incertain embodiments the pressure line 106 is a narrow line thatconstricts flow thereby reducing the response time of thepneumatic/hydraulic unit. A pressure controller 105 is optionallyincorporated into the pneumatic/hydraulic unit to regulate flow into andout of the pressure source and/or to regulate valves 306 and/or 304,and/or 302.

Any of the foregoing systems and devices can further include units toinduce an artificial arterial pressure pulse. Such units include, butare not limited to a linear actuator, as described above (see, e.g.,Maltz and Budinger supra.), a disk-type and a nozzle-flapper typeelectro-pneumatic converter (EPC) for the cuff-pressure control (see,e.g., Tanaka et al. supra), a standard cuff, and the like.

FIG. 9 provides flow chart illustrating typical acts performed in ameasurement of the effect of a stimulus on endothelial function. Thesubject is typically allowed to rest (e.g., for at least 1 minute, atleast 2 minutes, at least 3 minutes, at least 4 minutes, at least 5minutes, at least 10 minutes, at least 15 minutes, etc.) to avoid theeffect of transient activity of other stimulation on the measurement.The subject may be required to avoid eating, taking medicine, smokingand/or drinking coffee for certain periods of time (e.g., two hours ormore before the test). The cuff or cuffs (e.g., depending on whether atransit time calculation is to be made) are affixed to the desiredregions(s) of the subject (e.g., arm, leg, wrist, ankle, etc.). Theblood pressure of the subject is optionally determined using any methodknown in the art and/or using the system itself. The cuff(s) are theninflated to a substantially continuous pressure at or below the measureddiastolic pressure of the subject. Thus, in certain embodiment the cuffsare inflated to a pressure below the measured (or mean or medianmeasured) diastolic pressure (e.g., not less than about 10 mm Hg belowthe diastolic pressure, or not less than about 15 mm Hg below thediastolic pressure, or not less than about 20 mm Hg below the diastolicpressure, or not less than about 25 mm Hg below the diastolic pressure,or not less than about 30 mm Hg below the diastolic pressure). Apressure pulse or series of pressure pulses resulting from one or morecardiac cycles is then recorded providing baseline pressure versus timedata. The data is optionally processed to provide one or more parameters(e.g., maximum expansion, integrated pressure/time, maximum slope ofpressure pulse, transit time of pulse from one cuff to a second cuff,etc.).

A stimulus is then applied to the subject. Any of a number of stimuliexpected to alter endothelial function are contemplated. Such stimuliinclude, for example, occlusion of blood flow, and/or application of oneor more drugs to the subject. Illustrative drugs include, for example,drugs that act as NO agonists (e.g. acetylcholine), β-adrenergicagonists such as albuterol, acoustic/mechanical tissue vibration,transcutaneous low frequency ultrasound (see, e.g., Iida et al. (2006)J.Amer. Coll. Cardiol., 48(3): 532-537), and the like. The contribution ofbasal NO release to basal vascular tone may be elicited by administeringNO-synthase inhibitors such as L-NMMA and L-NAME. These agents may beadministered via intra-arterial infusion (as is conventional practice)or by means of novel administration methods we have demonstratedinvolving nasal inhalation and ingestion. Endothelium-independent smoothmuscle function may be evaluated by administration of NO-releasing drugssuch as nitroglycerin and sodium nitroprusside.

In certain embodiments, the stimulus excludes occlusion and/orapplication of drugs. In certain embodiments the stimulus excludesocclusion and/or application of drugs that are NO agonists.

In certain embodiments the stimulus comprises acoustic/mechanical tissuevibration, or transcutaneous low frequency ultrasound.

A pressure pulse or a series of pressure pulses resulting from one ormore cardiac cycles is then recorded providing stimulus-effectedpressure versus time data. The data is again optionally processed toprovide one or more parameters (e.g., maximum expansion, integratedpressure/time, maximum slope of pressure pulse, transit time of pulsefrom one cuff to a second cuff, etc.).

The baseline data or derived parameters is then compared to thestimulus-effected data or derived parameters to determine the presence,absence, and/or magnitude of the effect of the stimulus. In certainembodiments the results may be recorded in a database (e.g., in amedical record).

In certain embodiments the blood pressure can be eliminated and thecuffs simply inflated to a predetermined or arbitrary substantiallyconstant pressure.

In certain embodiments when using occlusion as a stimulus, Alternativelyto occluding the same artery on which the measurements are performed, adifferent artery connected to the measured artery, is occluded. Forexample, when the measurements are performed on the brachial artery, theocclusion may be applied to the radial and/or ulnar arteries. Ideally,when such a cuff is used to assess endothelial function, the occludingcuff is placed downstream of the points of measurement. This increasesthe contribution of NO-dependent mechanisms to the vasodilation thatoccurs, and minimizes the effects of tissue ischemia (which, aresubstantially mediated by other biochemical pathways not dependent onNO). The two cuffs may be integrated into a single entity containing twofillable air cavities. The upstream cavity is inflated only during themeasurement intervals (to subdiastolic pressures), while the downstreamcavity is used only for inducing endothelial stimulus via reactivehyperemia (inflated to suprasystolic pressures). In this way, themeasurement is always obtained in an arterial segment that was notsubject to ischemia.

The baseline phase measurement(s) optionally includes a plurality ofrounds (e.g., 2-5 rounds), in each of which the pressure versus timedata are recorded. The results of the plurality of measurement roundscan be optionally averaged to, in principle, reduce noise in themeasurements. In addition to, or as an alternative, other noise reducingstatistical methods can be utilized. Alternatively, in certainembodiments a single measurement is performed in order to limit the timerequired for the measurement session. Several of the earliest baselinemeasurement rounds may be discarded according to a predeterminedprotocol in order to minimize any initial deformation of the limb crosssection that may occur during the first measurements.

In certain embodiments the stimulus-effected measurements are made apredetermined time after application of the stimulus, e.g., when thestimulus effect is expected to be maximal.

In various embodiments repeated measurement rounds can be made afterperiods of reduced or eliminated cuff pressure to prevent the repeatedmeasurement rounds from inducing hyperemia which would influence themeasurements and/or prevents the repeated measurement rounds fromcausing discomfort to the patient.

As indicated above, in certain embodiments a score or derived parameterrepresentative of endothelial function is determined based on the effect(or absence of effect) of the stimulus (depending on the stimulus used).In certain embodiments the score is compared to a threshold andaccordingly a binary diagnosis is provided (e.g., normal, abnormal). Insome embodiments, the threshold depends on one or more attributes of thepatient, such as gender, height, weight and/or age. Alternatively oradditionally, a multi-level diagnosis is provided, for example giving avalue in percentages or other units. The multi-level diagnosis isoptionally determined by comparing the score to an array of thresholdsor to a “standard” curve.

As mentioned above, during the test session, between the base line phaseand the stimulus affected measurement, the subject preferably remains atrest, so as to minimize the difference in conditions between themeasurements. Alternatively or additionally, the results are correctedfor changes in the conditions between the phases.

As indicated above, in some embodiments, the difference in the baselineand stimulus effected parameters is calculated by determining anenvelope of the measurements and finding a maximum value with theenvelope to use as the basis of the parameter calculation. In certainembodiments the maximal difference in the value of the parameter(s)between the baseline and stimulus-effected parameters is determined. Thecalculation is performed using any method known in the art, such asusing a fitting method which finds a maximal difference over a singlecardiac cycle, or over a plurality of cardiac cycles (e.g., 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or more cardiac cycles).

As mentioned above, the systems described herein can be used todetermine the blood pressure of the subject patient, during endothelialfunction tests or separately. Typically, such measurements can be madeby inflating the cuff to a pressure above the systolic pressure of thesubject and the air pressure of the cuff is deflated to below thediastolic blood pressure of the patient. During the air pressuredeflation, pressure transducer 102 registers the changes in the pressureof measurement cuff 100. The resulting data are then analyzed to findthe systole (SYS), and/or diastole (DIA) pressures, using any of themethods known in the art for oscillatory blood pressure measurement.

It is noted that a poor dilatation functioning may occur due toarteriosclerosis of a specific artery. In order to preventidentification of endothelial dysfunction in subjects that have localarteriosclerosis in a single artery but do not suffer from endothelialdysfunction, in some embodiments the methods described herein arerepeated on another artery of the subject, for example on the oppositearm. If dysfunction identified for one artery but not the other, thesubject is identified as not having endothelial dysfunction and/or issent for additional tests.

In some embodiments, the microprocessor 206 and/or separate computer isprogrammed to carry out a complete test session automatically withoutrequiring instructions from a human operator. Optionally, control unit200 checks that the conditions are proper and stops the test session ifa problem is detected, for example when signal is detected, whenpressure exceeds a threshold, or when no sensible data is producedperhaps due to erratic or significant oscillations in the blood pressureof the subject during the test.

Alternatively, the operation sequence of a test session may be partiallyor entirely human operated. For example, each measurement phase may becontrolled automatically by microprocessor 206, while the initiation ofeach phase is controlled by a human operator. Optionally, an operatormay program operation sequences through a computer or other device.Alternatively or additionally, required operation sequences arepreprogrammed into microprocessor 206 at the time of manufacture.

Practical Low Cost Systems.

In certain embodiments the prototype illustrated in FIG. 1 may use anexpensive and bulky pneumatic regulator to produce constant pressure inthe cuff during measurement. In contrast, one illustrative and lessexpensive portable prototype is shown in the schematic diagram of FIG.12, and photographs of FIG. 13. This illustrative, but non-limitingembodiment uses a miniature pump and solenoid valve to control cuffpressure. Since the pump and solenoid valve provide on-off control, thepressure in the cuff generally falls with time as the tissue under thecuff displaces. There are a number of compelling reasons to use on-offcontrol: 1) There is no need for an expensive pressure regulator andcompressed air source; 2) The pump preferably does not operate duringmeasurement as it introduces noise into the signal; 3) The componentcount is smaller and the cost is much lower; and 4) Standard pumps andvalves employed in home blood pressure measurement systems can be used.Since the pump and valve may be actuated during a measurement interval,the recorded signal may be contaminated with noise. For offlineprocessing applications, this can be removed using low-pass filtering ofthe recorded time-series. For online processing, the times of actuationcan be fed into a data analysis algorithm to ensure this noise does notconfound the analysis.

Data Analysis for On-Off Systems.

To address this issue, a method of data analysis was developed thatimproves the accuracy of a system with on-off control to the extent thatimpressive results such as those shown in Table 2 are possible. ConsiderFIG. 15, which demonstrates the typical fall in pressure during ameasurement interval. The curves in FIG. 16 illustrate the effect ofarticle unloading on arterial compliance (Bank et al. (1999)Circulation, 100: 41-47). A decrease in unloading pressure of 8 mm Hg(as seen in FIG. 15) can impact compliance significantly when thetransmural pressure is small (10-20 mm Hg in our case at diastole).Clearly, when comparing pulse properties such as amplitude and maximumupward slope, before and after stimulus, it is preferably to comparepulses measured at like cuff pressures. For example, in FIG. 15, whileit is appropriate to directly compare the post-stimulus and baseline att=20 s, this is not the case at t=10 s. In the former case, the cuffmeasurement pressure is similar, but in the latter case, it is largerduring the post-stimulus series than during the baseline series.

One illustrative approach to this issue is to “histogram” the pulses bypressure, using a binning statistic such as the mean, median, minimum ormaximum pressure during the pulse. Pulses in each histogram bin from thebaseline and response series are compared and the fractional change iscomputed for each bin. A weighted average of the bins is taken, wherethe weights are proportional to the number of pulses in each bin and theconfidence in each measurement.

In cases where the ranges of pressure do not completely overlap, curvessuch as those shown in FIG. 16 can be used to adjust the data so allpulses can be compared.

Another method by which one may perform the analysis is to applyregression on the pressure characteristic during the measurement period.The regression curve is then used to scale the individual pulses to acertain reference pressure. Pulses obtained at different pressure valuescan be compared as if these were all obtained at the reference pressure.

Increasing Measurement Pressure Stability.

As described above, regression analysis can be applied to address theproblem of mean measurement pressure variation following initialattainment of a pressure set point. Two additional schemes are proposedto address this issue without requiring a pneumatic feedback system suchas a pressure regulator.

As is illustrated in FIG. 17, divide each of the measurement intervals Tcan be divided into two segments, T₁ and T₂, such that T=T₁+T₂. Thepurpose of T₁ is to stabilize the pressure close to the measurementpressure set point during the period where tissue compression under thecuff leads to a natural pressure drop. Once the pressure has stabilized,or otherwise, T₂ begins, during which no control of the pressure isexercised, or the criteria for initiating pressure corrections areconsiderably relaxed.

In one illustrative scheme, we substitute the solenoid on-off deflationvalve in FIG. 12 with a proportional valve as illustrated in FIG. 14. Atypical example of a miniature proportional valve is the VSO-MI (ParkerHannifin Corp., Cleveland Ohio). During T₁, the pump continues to run,while a control feedback system adjusts the proportional valve such thatthe desired pressure set point is maintained. An advantage of thisscheme is that it is not necessary to adjust the pump output, which maynot be feasible with many pump types. During T₂, either:

1) The pump is deactivated and the valve is shut fast; or

2) A typical on-off servo control regime is implemented.

The second scheme relies on assigning different pressure tolerances toT₁ and T₂, namely ΔP₁ and ΔP₂. During interval T_(n), adjustment of thepressure is only initiated when the cuff pressure P<P_(s)−ΔP_(n) orP>P_(s)+ΔP_(n) wherein P_(s) is the measurement pressure set point.Thus, for example, during interval T₁, the pressure tolerance (P_(t)) isP_(t)=P_(s)±ΔP₁, while during interval T₂, the pressure tolerance isP_(t)=P_(s)±ΔP₂. When the actual cuff pressure P goes above or below therange for P_(t) the pressure is adjusted. By setting, for example,ΔP₂>ΔP₁, it is possible to avoid unnecessary servoing during T₂ that mayrender measurement data unusable. FIG. 17 provides one illustrative, butnon-limiting example of these ranges, and the interpretation of thesequantities.

Apparatus to provide uFMD measurements.

In various embodiments an apparatus for assessment of endothelialfunction in a mammal is proved where the apparatus can generate uFMDresults. In certain embodiments the apparatus comprises a measurementcuff adapted to apply a substantially pressure to an artery in saidmammal; a measurement unit adapted to detect and quantify over one ormore cardiac cycles, pressure pulses in said cuff while said pressure isapplied; a controller that is adapted to apply to the cuff a differentpressures within a single time period (Tm), and/or to applysubstantially constant pressures that differ in different time periods(T_(m)) where the controller monitors and adjusts the pressure in thecuff and whose response time is sufficient slow so that the changes inpressure resulting from said cardiac cycles are not substantiallyattenuated by said system, and/or that is adapted to control a pressuresource and a valve to provide on-off control of the pressure in saidcuff; and a processor adapted to determine a plurality of baselinevalues for a parameter related to endothelial function as a function ofapplied pressure (P_(m)) and to determine a plurality ofstimulus-effected values for a parameter related to endothelial functionas a function of applied pressure (P_(m)).

In certain embodiments the processor is adapted to determine the area ofthe arterial lumen calculated from the baseline pressure measurements asa function of transmural pressure P_(tm), where transmural pressure isdetermined as the difference between the baseline measured pressures andthe external cuff pressure P_(m) and fitting these data with a firstnon-linear model to provide a first function describing baselinearterial lumen area as function of transmural pressure; to determine thearea of the arterial lumen calculated from the stimulus-effectedpressure measurements as a function of transmural pressure P_(tm), wheretransmural pressure is determined as the difference between the measuredstimulus-effected pressures and the external cuff pressure P_(m) andfitting these data with a second non-linear model to provide a secondfunction describing stimulus-effected arterial lumen area as function oftransmural pressure; to use the first function to calculate baselinearterial lumen area (A_(b)) at a transmural pressure substantially equalto the systolic blood pressure; and to use the second function tocalculate stimulus-effected arterial lumen area (A_(r)) at a transmuralpressure substantially equal to the systolic blood pressure. In certainembodiments the processor is configured to determine the equivalentultrasound-based FMD (uFMD) where uFMD is proportional to the squareroot of the ratio A_(r)/A_(b). In certain embodiments the processor isconfigured to determine the equivalent ultrasound-based FMD is given as

uFMD%=[√{square root over ((A _(r) /A _(b))}−1]×100

In certain embodiments the first non-linear model and/or said secondnon-linear model are the same type of function. In certain embodimentsthe first non-linear model and/or said second non-linear model areselected from the group consisting of a 2 parameter model, a 3 parametermodel, a four parameter model, a 5 parameter model, and a six parametermodel. In certain embodiments the first non-linear model and said secondnon-linear model are both arctangent models.

In certain embodiments the processor is configured to determine thepressure in the cuff as a function of time to provide a cardiac pulsewaveform while the cuff inflates and/or while said cuff deflates. Incertain embodiments the processor is configured to compare cardiac pulsewaveforms between baseline and post-stimulus conditions either by directcomparison of pulse characteristics at corresponding pressure levels orby first fitting models to the set of baseline cardiac pulse wave formsand to the set of post-stimulus cardiac pulse waveforms and comparingparameters generated by the two models.

In certain embodiments the apparatus is configured to measure bloodpressure and cFMD simultaneously. In certain embodiments the apparatusis configured to measure the systolic BP and MAP and to calculate thediastolic blood pressure (DBP) using an oscillometric analysis; and/orthe apparatus is configured to measure the systolic BP and MAP aremeasured and to determine the DBP by analyzing Korotkoff sounds obtainedusing an audio sensor (e.g., stethoscope, phonocadiogram) or ultrasoundprobe. In certain embodiments the apparatus is configured to calculatecFMD and/or MAP, and/or DBP from measurements obtained during cuffinflation; and/or the apparatus is configured to calculate cFMD and/orMAP, and/or DBP from measurements obtained during cuff deflation. Incertain embodiments the apparatus is configured to determine systolic BPand/or diastolic BP and, for cFMD determination, to provide a comparisonof pulse waveform characteristics between baseline and post-stimulusintervals at transmural pressures calculated using the determinedsystolic and/or diastolic BP values.

In certain embodiments the apparatus is configured to perform the methodfor determining uFMD as described herein, except application of thestimulus.

In certain embodiments the apparatus is configured to perform the methodfor determining uFMD as described herein, including application of thestimulus where the stimulus comprises restricting flow of blood to thelimb by occlusion of a blood vessel. In certain embodiments theapparatus is configured to restricting the flow of blood using a cuff.In certain embodiments the apparatus is configured to restrict the flowof blood and applying the pressure on the artery are using separatecuffs. In certain embodiments the apparatus is configured to use thesame cuff is used to occlude the blood vessel and to apply the pressureon the artery. In certain embodiments the apparatus is configured toinflate a restricting cuff to a pressure at least 10 mm Hg abovemeasured systolic blood pressure for the mammal. In certain embodimentsthe apparatus is configured to restricting flow of blood through theartery for at least 1 minute.

In certain embodiments the controller is configured to apply pressure tothe cuff during one or more time periods (T_(m)) by adjusting a pump orother pressure source and/or a proportional release valve to maintain adesired pressure. In certain embodiments the controller is configured tostop adjustment of said pressure during the time period(s). In certainembodiments the controller is configured to periodically adjust saidpressure using an on-off control system during the time period(s). Incertain embodiments the controller is configured to maintain pressurewithin a pressure range (AP) around a measurement set point during atime period. In certain embodiments the pressure range (AP) ranges fromabout 1 mm Hg to about 6 mm Hg, or from about 1 mm Hg to about 4 mm Hg,or from about 1 mm Hg to about 3 mm Hg, or from about 1 mm Hg to about 2mm Hg. In certain embodiments the apparatus is configured to provide atime interval duration ranging from about 1 sec, or from about 2 sec, orfrom about 3 sec, or from about 4 sec, or from about 5 sec, or fromabout 6 sec, or from about 7 sec, or from about 8 sec, or from about 9sec, or from about 10 sec, or from about 15 sec up to about 20 sec, orup to about 30 sec or up to about 40 sec or up to about 50 sec, or up toabout 1 min, or up to about 2 min, or up to about 3 min, or up to about4 min, or up to about 5 min, or up to about 6 min, or up to about 7 min,or up to about 8 min, or up to about 9 min, or up to about 10 min, or upto about 15 min, or up to about 20 min, or up to about 25 min, or up toabout 30 min.

In various embodiments the controller is configured to monitor andadjust said pressure at a response time sufficiently slow so that saidpressure changes resulting from said cardiac activity are attenuated byless than 10%. In certain embodiments the controller is configured tomaintain the external pressure by setting the pressure in a cuff to avalue and not altering external pressure applied to the cuff during themeasurements of pressure variations due to said cardiac activity. Incertain embodiments the controller is configured to apply an externalpressure equivalent to or below a diastolic pressure determined for saidsubject. In certain embodiments the controller is configured to apply anexternal pressure below the average diastolic pressure measured for thesubject or below an expected diastolic pressure for the subject. Incertain embodiments the controller is configured to apply an externalpressure below the average diastolic pressure measured for said mammal,but no less than about 10 mmHg below said average diastolic pressure. Incertain embodiments the controller is configured to apply a constantpressure at different levels during different measurement intervals.

In various embodiments the apparatus comprises a hydraulic or pneumaticpump adapted to apply the pressure to one or more cuffs. In certainembodiments the response time is reduced by disposing a narrow pressureline between hydraulic or pneumatic pump and a cuff. In certainembodiments the apparatus comprises a valve and a pump configured toprovide on-off control of the pressure in one or more cuffs. In certainembodiments the apparatus further comprises an accelerometer disposed todetect movement or vibrations in said cuff or apparatus.

In various embodiments the cuff is pressurized with a material selectedfrom the group consisting of a gas, a fluid, and a gel. In variousembodiments the cuff is adapted to apply pressure substantially aroundan entire circumference of a limb including the artery. In certainembodiments the cuff is adapted to apply a local pressure that does notsubstantially affect other blood vessels in a same limb as the artery.

In various embodiments the processor in the apparatus is configured todetermine a blood pressure. In various embodiments the processor isconfigured to calculate the external pressure to be applied based on oneor more blood pressure measurements and to direct the controller toapply the calculated external pressure. In certain embodiments thecontroller is configured to induce at least one of measurement roundresponsive to an indication that a stimulus was administered to theartery and at least one of the measurement rounds before the indicationthat the stimulus was administered to the artery is received.

In certain embodiments the controller is adapted to apply the pressurecontinuously over at least five cardiac cycles of the patient. Incertain embodiments the controller is configured to store over thecourse of one or more cardiac cycles, changes in pressure in said cuffresulting from cardiac activity of the mammal as a function of time.

In certain embodiments the processor is configured to integrate thevalue of a pressure change over time (calculate the area under apressure/time curve) for one or for a plurality of cardiac cycles todetermine an integrated pressure value.

In certain embodiments the processor is configured to determine themaximum of the derivative of the pressure versus time wave form on therising edge of a pressure pulse for one or for a plurality of cardiaccycles to determine a compliance value. In certain embodiments theprocessor is configured to average said integrated pressure value and/orsaid compliance value over a plurality of cardiac cycles. In certainembodiments the processor is configured to determine the integratedpressure value and/or the compliance value for a single cardiac cycle.In certain embodiments the processor is configured to determine saidintegrated pressure value and/or said compliance value and identify amaximum change in said value between a baseline measurement and astimulus-effected measurement.

Subject Motion

From our human subject studies, it is apparent that oscillatory subjectmotion such leg shaking can introduce spurious waveforms that may beinterpreted as pulses. This can be addressed by means of software and/orhardware. One software approach is to perform real-time analysis of theincoming pressure signal and detect anomalies. In a hardware approach,an accelerometer can be placed on the cuff, on the cuff tube or in theinstrument itself to detect vibrations that cannot be easily filteredout (e.g., those that are in the same frequency band as the signal ofinterest). The system can then generate an alert to the user indicatethat vibration is present and may abort the measurement if vibrationdoes not cease.

It will be appreciated that the above described methods and apparatusmay be varied in many ways, including, changing the order of acts of themethods, and the exact implementation used for the apparatus. It shouldalso be appreciated that the above described methods and apparatus areto be interpreted as including apparatus for carrying out the methodsand methods of using the apparatus.

The devices and methods have been described herein using non-limitingdetailed descriptions of embodiments thereof that are provided by way ofexample and are not intended to limit the scope of the invention. Forexample, rather than performing the endothelial dysfunction test on thearm, the method may be performed on a subject's leg.

In addition, while the methods are described with reference to humans,the term mammal is intended to include humans as well as non-humanmammals (e.g., non-human primates, canines, equines, felines, porcines,bovines, ungulates, largomorphs, and the like).

Home Health Monitoring.

In certain embodiments the methods and devices described herein are wellsuited to home health monitoring. In certain embodiments the devicesdescribed herein can be provided as off-the-shelf products comprising a1-cuff or a 2-cuff system, and typically, although not necessarily acontrol system to operate the cuffs. The device is typically configuredto executes one or more measurement protocol(s) (e.g., coordinatedsequence of inflations, pressure holds, pressure measurements, restperiods and deflations).

In certain embodiments (e.g., as illustrated in FIG. 17 the system canlink to a computer, a tablet, and/or a cell phone, e.g., via a directcable, via a wireless (e.g., wifi) link, or via a Bluetooth). In certainembodiments the computer/tablet/cell phone runs an application (e.g., anapp) that allows specification of, e.g., measurement protocol parameters([measurement, rest, inflation durations], number of measurements,measurement and occlusion pressures, etc.). The computer/tablet/cellphone can receive raw pressure data or processed data from the cuff,optionally stores the data, and can send it to a server via theInternet.

The server (e.g., provided by a healthcare providing or healthmonitoring service) can performs (offline) analysis of raw pressure (orprocessed) data using our algorithms. The server/service can preparereports and email links to results to investigators or home users orprovides access via webpage.

The server can optionally integrate with other web-based health and/orfitness monitoring services provided by the same or other providers.Such services monitor data such as weight/weight change, heart rate,blood pressure, blood sugar, exercise level, and the like. In certainembodiments, the data can be integrated into a medical record for thesubject where the medical record is maintained by a health careprovider, and/or an insurance provider, and/or a physician or otherhealthcare provider, and/or a web-based healthcare monitoring service,and/or the user. Illustrative healthcare monitoring services include,but are not limited to FITBIT®, WITHINGS®, SYNCMETRICS®, ROCKHEALTH®,HEALTHBOX®, DREAMIT HEALTH®, NY DIGITAL HEALTH ACCELERATOR®, and thelike.

Use of Measures of Endothelial Function in a Clinical Context.

As explained above, devices and methods are provided herein for theassessment of endothelial function in a mammal (e.g., in a human).Although studies often report endothelial dysfunction as a loss of thevasodilatory capacity (in response to a NO stimulus, likeacetylcholine), the term encompasses a generalized defect in many or allthe homeostatic mechanisms maintaining/modulating vascular homeostasis.Endothelial dysfunction is a broad term that can imply diminishedproduction of or availability of NO and/or an imbalance in the relativecontribution of endothelium-derived relaxing and contracting factors(such as ET-1, angiotensin, and oxidants).

For example, endothelial dysfunction in diabetes may result from adecreased bioavailability of NO (secondary to insulin resistance)coupled with an exaggerated production of ET-1 (stimulated byhyperinsulinemia or hyperglycemia). Endothelial dysfunction has beenimplicated in the pathogenesis and clinical course of all knowncardiovascular diseases and is associated with future risk of adversecardiovascular events.

It is well accepted that endothelial dysfunction occurs in response tocardiovascular risk factors and precedes the development ofatherosclerosis (see, e.g., Shimokawa (1999) J. Mol. Cell Cardiol. 31:23-37; Ross (1999) N. Engl. J. Med. 340: 115-126). It is generally aprevailing paradigm that endothelial dysfunction can form a common linkbetween risk factors and atherosclerotic burden. Endothelial dysfunctionactively participates in the process of lesion formation by promotingthe early and late mechanisms of atherosclerosis. These includeupregulation of adhesion molecules, increased chemokine secretion andleukocyte adherence, increased cell permeability, enhanced LDLoxidation, platelet activation, cytokine elaboration, and vascularsmooth muscle cell proliferation and migration. Endothelial dysfunctionalso plays an important role in the clinical course of atherosclerosis.

Impaired endothelium-dependent vasodilatation in coronary arteries withestablished atherosclerosis results in paradoxical vasoconstriction,which may result in reduced myocardial perfusion and myocardialischemia. Additionally, endothelial dysfunction actively modulatesplaque architecture and portends the vulnerability of the lesion and thelikelihood of rupture. Through this vasoconstrictor and inflammatorymechanism, endothelial dysfunction in atherosclerotic vessels may leadto the development of unstable coronary syndromes (see, e.g., Verma etal. (2002) Circulation, 105: 546-549).

Accordingly, in various embodiments the devices and methods providedherein for assessment of endothelial function can find utility in aclinical context. Thus, in certain embodiments, particularly in thecontext of a differential diagnosis, the measures of endothelialfunction provide by the devices and methods described herein findutility in the diagnosis and/or prognosis and/or in the treatment ofvarious cardiovascular pathologies. In certain embodiments the measureof endothelial function, particularly in the context of a differentialdiagnosis, is an indicator of a cardiovascular pathology (e.g.,atherosclerosis, hypercholesterolemia, high LDL-C, low HDL-C, highlipoprotein (a), small dense LDL-C, oxidized LDL-C, hypertension, highhomocysteine, aging, vasculitis, preeclampsia, metabolic syndrome,variant angina, diabetes, active smoking, passive smoking,ischemia-reperfusion, transplant atherosclerosis, cardiopulmonarybypass, postmenopausal, Kawasaki's disease, Chagas' disease, familyhistory CAD, infections, depression, inactivity, obesity, renal failure,increased CRP, congestive heart failure, left ventricular hypertrophy,etc.). In certain embodiments the measure of endothelial function insaid mammal is printed out and/or stored on a tangible medium which, incertain embodments can comprise a medical record (e.g. a computerizedmedical record, a paper medical record, a medical record chip or ID, andthe like).

Also, in certain embodiments methods of treating a subject for acardiovascular pathology are provided where the methods involveassessing endothelial function in the subject using the apparatus and/ormethods described herein and, where the method(s) produce a measureindicating impaired endothelial function, evaluating the measure in thecontext of a differential diagnosis for a cardiovascular pathology and,where indicated, performing one or more interventions for the treatmentof a cardiovascular pathology. In certain embodiments the cardiovascularpathology comprises a pathology selected from the group consisting ofatherosclerosis, hypercholesterolemia, high LDL-C, low HDL-C, highlipoprotein (a), small dense LDL-C, oxidized LDL-C, hypertension, highhomocysteine, aging, vasculitis, preeclampsia, metabolic syndrome,variant angina, diabetes, active smoking, passive smoking,ischemia-reperfusion, transplant atherosclerosis, cardiopulmonarybypass, postmenopausal, Kawasaki's disease, Chagas' disease, familyhistory CAD, infections, depression, inactivity, obesity, renal failure,increased CRP, congestive heart failure, and left ventricularhypertrophy. In certain embodiments the intervention comprises one ormore interventions selected from the group consisting of prescribingand/or administering ACE inhibitors, prescribing and/or administeringangiotensin receptor blockers, prescribing and/or administeringendothelin blockers, prescribing and/or administering statins,prescribing and/or administering tetrahydrobiopterin, prescribing and/oradministering folates, improving insulin sensitivity, LDL reduction, HDLaugmentation, prescribing and/or administering antioxidants, prescribingand/or administering estrogen, prescribing and/or administeringL-arginine, prescribing and/or administering desferoxamine, prescribingand/or administering glutathione, homocysteine reduction, lowering CRP,and reducing free fatty acid flux.

It should be understood that the methods and apparatus described hereinmeasure endothelial dysfunction by means of measuring the consequencesof vascular smooth muscle relaxation, and that these methods maytherefore be applied to measure smooth muscle function simply bysubstituting the endogeneous source of nitric oxide (endothelial NOrelease) with an exogenous source, such as sublingual nitroglycerin.

It should be understood that features and/or steps described withrespect to one embodiment may be used with other embodiments and thatnot all embodiments of the invention have all of the features and/orsteps shown in a particular figure or described with respect to one ofthe embodiments. Variations of embodiments described will occur topersons of the art.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Validation of Measurement of Endothelial Function

One way to determine the value of a new measure is to determine to whatdegree it is correlated to a “gold standard” measurement. In the case ofendothelial function, the gold standard is dilation of coronary arteriesin response to infused acetylcholine (ACh). This procedure is invasive,expensive and suitable only for diseased patients undergoing cardiaccatheterization.

In the evaluation of a previous instrument that we developed for theassessment of endothelial function, we determined the correlationbetween our method and ultrasound-based FMD measurements in the brachialartery. While non-invasive, FMD studies are technically difficult andproduce measurements with high variance.

As described herein , it is believed that that such studies areunnecessary in order to make a determination that a method is effectivein assessing endothelial function. Physical methods for arterial EFevaluation typically measure changes in the material properties of theartery. The changes that occur in response to endogenous release of NOare similar in nature and magnitude to those that occur followingadministration of exogenous NO via agents such as nitroglycerin (NG). Asa consequence, if it is shown that a measurement method is sensitive tovasorelaxation induced by NG, it can be assumed that the technique willalso be sensitive to endothelium-mediated vasorelaxation. A majoradvantage of this method is that response to NG is intact even inindividuals with endothelial dysfunction, so there is no need to performa correlation analysis between two measurements.

To further strengthen the case, the measurement method shoulddemonstrate sensitivity to RH-induced vasorelaxation in individuals whowould be expected to have intact endothelial response.

Three individuals in the age-range of 28-38 were examined. Table 1 liststhe subject characteristics. All subjects had Framingham risk scores of1% or less, and had no history of cardiovascular disease. Eachindividual was assessed at least three times before and after RH inducedby five minutes of suprasystolic cuff occlusion. At least one additionalmeasurement was made using the same protocol, except without cuffinflation. Sensitivity to a 0.4 mg dose of sublingual NG was assessedthree times in two individuals.

TABLE 1 Subject characteristics. (NS: no stimulus, RH: reactivehyperemia, NG: nitroglycerin). Number of Framingham Studies SubjectGender Age Score NS/RH/NG Subject 1 Male 38 1% 3/6/3 Subject 2 Female 38<1% 4/3/3 Subject 3 Male 28 <1% 1/3/0

Each individual was assessed at least three times before and after RHinduced by five minutes of suprasystolic cuff occlusion. At least oneadditional measurement was made using the same protocol, except withoutcuff inflation.

Sensitivity to a 0.4 mg dose of sublingual NG was assessed three timesin two individuals. In the analysis that follows, all of the datasetsare pooled by stimulus. This addresses the question: Can the methodmeasure changes in arterial tone due to vasorelaxatory stimuli and withwhat sensitivity?

A single quantity relating to the recorded pressure data, the pulseamplitude, was studied and it was posited that this is proportional tothe arterial area. During the post stimulus interval, pressure data fromthe cuff were recorded approximately every 80 seconds, for a period of30 seconds. During each recording interval, the cuff was inflated to 70mm Hg, which was always below the measured diastolic pressure of thesubject. To quantify the observed response, the mean of the pulsequantity (in this case, amplitude) during the response interval wasdivided by the mean value of the same quantity during the baselineinterval. FIG. 10 illustrates the results.

Three experiments were also performed 3 experiments on Subject 1 wheredilation in the right arm was measured using ultrasound concurrentlywith dilation in the in the left arm measured using the device. Thepurpose of these studies was to examine the correlation of theresponse-vs-time curves.

Results

FIG. 10 is strongly supportive of the hypothesis that the method issensitive to smooth muscle relaxation for the following reasons: For theNG studies, in the time interval from 6 minutes to 20 minutes, there isa large and persistent difference between the NG responses and the NSresponses. For the RH studies, there is no overlap between the RH and NSdata responses during the four minutes following cuff release.

In Table 2, the maximum response for each stimulus is calculated and thestatistical significance of the change relative to the NS case (one-tailStudent's t test) is evaluated. Values of p<0:05 were consideredsignificant.

TABLE 2 Statistical analysis of amplitude response. Mean ± SEM ofmaximum p-value Stimulus Response over all data sets versus NS RH 1.51 ±0.052 1.19 × 10⁻⁵† NG 1.70 ± 0.036 6.25 × 10⁻⁶† NS 1.01 ± 0.068 N/A

FIGS. 11A-11C provide the results of the 3 studies where measured arterydiameter was measured using ultrasound and simultaneously volumedistention was measured by the method described herein. The time coursesof dilation are similar in all three cases. As observed in our otherstudies, the sensitivity of our method is≈5 times greater than diametermeasurements. The diameter measurements exhibit high variance owing tothe extreme sensitivity of the method to slight movement of thesubject's arm

Conclusion.

While the current sample size of three subjects is small, the use ofrepeated measurements per subject has demonstrated with greatstatistical certainty that the proposed measurement device is capable ofdetecting changes due to RH (p=1.19×10⁻⁵) and NG (p=6.25×10⁻⁶) in allsubjects on all occasions. This statistical analysis invalidates thenull hypothesis that RH or NG evoke equal responses to NS in this set ofsubjects. The fact that there is no overlap between NS and either of theresponse classes in FIG. 10 is a truly impressive result.

As discussed above, since NG response is intact in almost allindividuals, little is gained in examining a larger population. Theresults show that the sensitivity of the method is approximately 5 timesgreater than that of ultrasound-based imaging of arterial diameter inresponse to flow-mediated dilation (FMD due to RH). This is based on acomparison of the 51% mean maximum increase in pulse amplitude overbaseline versus the approximate 10% brachial artery diameter changerepresentative of an intact endothelial response in B-mode ultrasoundFMD studies in the literature.

Example 2 Measurement of Brachial Artery Endothelial Function Using aStandard Blood Pressure Cuff

This example presents method of measuring changes in the cross-sectionalarea of the brachial artery that requires neither relatively costly andbulky ultrasound equipment, nor any technical skill on the part of theoperator. Instead of ultrasound, a standard blood pressure cuff is usedto take the measurement. The cuff is partially inflated during themeasurement process, so that changes in the area and compliance of thevessel can be calculated from tiny pressure variations in the cuff. Thepartially inflated cuff removes (mechanically unloads) stress from thearterial wall, and this amplifies the absolute change in area andcompliance seen in response to endothelial stimulus (Bank et al. (1995)Circ. Res. 77(5): 1008-1016), allowing ensuing vasorelaxation to bemeasured much more easily. The same cuff may be used to occlude theartery and thus provide reactive hyperemic stimulus for FMDmeasurements.

We begin by explaining the physical and physiological basis of themeasurement. We then describe the initial prototype of the device, andthen demonstrate that the device may be realized by reprogramming aconsumer-oriented electronic sphygmomanometer. The method is thenevaluated on human volunteers and the results are compared toultrasound-based FMD (uFMD) studies performed on the same limb 10minutes following the proposed cuff FMD (cFMD) measurements. A list ofabbreviations used in this paper appears in Table 3.

TABLE 3 Abbreviations used in this example. ACE angiotensin-convertingenzyme A/D analog-to-digital BP blood pressure CAD coronary arterydisease cD vasodilation due to any stimulus, measured using cuff-basedmethod cFMD flow-mediated vasodilation, measured using cuff-based methodCVD cardiovascular disease DC direct current (mean signal value) EDHFendothelium-derived hyperpolarizing factor EFMA endothelial function inmajor arteries FMD flow-mediated vasodilation NG Nitroglycerin NO nitricoxide NOS nitric oxide synthase NS no stimulus applied PC personalcomputer RH reactive hyperemia RH5 reactive hyperemia after release of 5minute occlusion SD standard deviation SEM standard error of the meanuFMD FMD, measured using ultrasound imaging

Methods.

2.1. Principles of Operation

One key to making FMD much easier to assess is to use a cuff to measurechanges in arterial cross-sectional area, instead of using ultrasoundimaging to measure arterial diameter. This allows us to eventuallycreate a subject-operated consumer-oriented measurement device that cantake advantage of convenient hardware and software platforms, such assmart phones and tablets, as we will describe in Section 2.3.3.

When the cuff is partially inflated so that it fits the arm snugly,changes in cuff pressure are proportional to changes in the volume ofthe underlying arm (this is the basic principle of plethysmography).Since blood volume changes most rapidly in the conduit arteries, therising edge of each pulse (diastole to systole) reflects changes in thevolume of these arteries enclosed by the cuff.

FMD studies seek to measure the amount of vascular smooth musclerelaxation that occurs as a consequence of endothelial stimulus. Thefundamental quantity affected by this relaxation is arterial wallcompliance (uFMD measures change in vessel caliber, which is only oneconsequence of relaxation of vascular smooth muscle (Nichols andO'Rourke, McDonald's blood flow in arteries, 3rd ed. Edward Arnold,1990, pp. 100-101]). We now explain how we can use the volume changemeasurements derived from the cuff to measure compliance.

The induction of local reactive hyperemia by means of cuff occlusion andsubsequent release does not change systemic blood pressure. Under thesecircumstances (which should ideally be verified for each study), thepressure changes observed from diastole to systole are proportional tothe concomitant volume changes. Let ΔV_(b) and ΔV_(r) denote the volumechanges from diastole to systole under baseline and post-stimulusresponse conditions. Since the cuff is part of a sealed pneumaticsystem, the pressure-volume product is constant (P V=k). If the cuffsnugly encloses the limb and the outer cuff sheath is non-elastic, thetotal volume (the volume of the enclosed limb+the volume of the cuff)maintains a constant value even as the blood volume changes. Anincrement in arterial pressure leads to an increase in arterial volume,which reduces the volume of the cuff by an equal amount (by compressingits contents). This, in turn, effects a pressure increase in the cuffthat is proportional to the volume change in the artery.

Stating this formally:

V _(l) =V _(c) =V _(total)=(V _(l) −ΔV)+(V _(c) +ΔV) and

P _(c) V _(c) =k=(P _(c) +ΔP)(V _(c) +ΔV),

where Pc is the cuff pressure, Vc is the cuff volume and ΔV is thechange in volume of the enclosed limb, V₁. We now solve for the observedchange in cuff pressure ΔP as:

$\begin{matrix}{{\Delta \; P} = {{- \frac{P_{c}}{V_{c} - {\Delta \; V}}}\Delta \; {V.}}} & (1)\end{matrix}$

This is non-linear in ΔV, but since we have ΔV<<Vc (the perturbation inthe cuff volume due to the pulse is much smaller than the cuff volume),this strongly approximates a linear relationship with a slope−P_(c)/V_(c). Since the length of the artery under the cuff, 1, does notchange appreciably during the cardiac cycle, we may thus assume thatΔP∝ΔA , where A is the cross-sectional area of the arterial lumen. If wedenote the pre- and post-stimulus areas as A_(b)=V_(b)/l andA_(r)=V_(r)/l, respectively, the cFMD metric is given by:

$\begin{matrix}{{{cFMD}\mspace{14mu} \%} = {\left\lbrack {\frac{A_{r}}{A_{b}} - 1} \right\rbrack \times 100.}} & (2)\end{matrix}$

This expression is an area analog of the standard FMD metric:

$\begin{matrix}{{{{uFMD}\mspace{14mu} \%} = {\left\lbrack {\frac{d_{r}}{d_{b}} - 1} \right\rbrack \times 100}},} & (3)\end{matrix}$

where d represents arterial diameter. It is important to remember thatthat the areas are obtained during wall unloading, and are not, ingeneral, equal to πd 2/4 (under the assumption of a circular crosssection), since those diameters are measured at full transmuralpressure.

The small volume changes that occur in the artery lead to very smallpressure changes in the cuff, which are difficult to measure accurately.However, as the degree of cuff inflation increases and more pressure isapplied to the limb, mechanical stress on the wall of the artery isrelieved by the cuff. This mechanical unloading decreases the influenceof stiff collagen fibers on the vessel wall properties, and this leadsto a large increase in vessel distensibility (Bank et al. (1996)Circulation, 94(12): 3263-3270).

FIG. 2 illustrates diametric distension waveforms obtained using M-modewall tracking (Wall Track System II, Pie Medical, Maastricht,Netherlands). Decreasing the transmural pressure by 80 mmHg leads to amore than twenty-fold increase in maximum distension in response to thesame diastolic to systolic pressure transition. This is consistent withthe very carefully executed intra-arterial ultrasound measurements ofBank and co-workers (Bank et al. (1995) Circ. Res. 77(5): 1008-1016).FIG. 16 illustrates the results of those studies, showing the change inbrachial artery compliance across the full range of transmural pressure.The compliance characteristic is shown before and after the arterialsmooth muscle is relaxed using nitroglycerin (NG). When the transmuralpressure is reduced to ≈25 mmHg, we see that the absolute difference invessel compliance between the baseline and relaxed state is maximized.The relevant observation is that relaxation of the artery (such as thatdue to FMD) is much easier to measure when the artery wall is unloaded,simply because the magnitude of the induced change is a larger quantity.A larger change in compliance means that a larger increase in arterialcross-sectional area is achieved for a given pressure rise from diastoleto systole.

In the above theoretical justification of the proposed measurementmethod, we assume that the tissue between the cuff and artery isincompressible, and that it does not change in volume between the pre-and post-stimulus intervals. The thickness and consistency of thistissue will affect the relationship between the volume of the artery andthe pressure in the cuff. However, since the cFMD metric is normalizedto a baseline measurement, as long as this relationship does not changebetween the pre- and post-stimulus measurement intervals, thecharacteristics of this tissue should not influence the results. It isreasonable to expect that the vasodilatory stimulus will cause somevasodilation of resistance vessels in the surrounding tissue, andelsewhere in the limb distal to the occlusion (Nichols and O'Rourke(1998) McDonald's blood flow in arteries, 4th ed. Edward Arnold, pp.258-259). The former effect will cause the cFMD metric to somewhatoverestimate the pure arterial response. The effect of the latter is todecrease wave reflection at distal sites (owing to arteriolar dilation),and this may reduce the amplitude of the systolic peak, leading tounderestimation of the arterial dilation. Since the rising edge of thedistension waveform (luminal volume) is in phase with the pressurewaveform (Meinders and Hoeks (2004) Ultrasound in Med. & Biol. 30(2):147-154), changes in wave reflection in the distal limb will bias bothuFMD and cFMD to a similar extent. We consequently can ignore thiseffect as a differential confounding influence.

To quantify the effect of vasodilation in intervening tissues, wecompare the 5%-95% rise times of the distension waveform (obtained usingM-mode wall tracking, as was used to produce the waveforms in FIG. 2)with the cuff pressure waveform. Similar rise times would imply thatthis part of the cuff pressure waveform (from which the cFMD metric ischiefly derived) is due to the direct effect of arterial luminal areaincrease. The reason for this is that low caliber collateral vesselsprovide much larger resistance to flow than conduit vessels and the timeconstant for volume change is thus much longer. For example, in thehuman finger, the pulse transit time over the short distance from thedigital arteries to the skin of the same finger is more than 200 ms,which is longer than the rise times of both the distension and cuffpressure waveforms (Bernjak and Stefanovska (2009) Physiol. Meas. 30(3):245). Examining 55 typical rising edges of the cuff pulse pressurewaveform, we calculate a mean (±SD) rise time of 133±8 ms. Thecorresponding distension mean rise time is 122±2 ms. Since the thicknessof the intervening tissue bed is much larger than that encountered inthe finger, it is unlikely that the volume change in the resistance bedcould appreciably contribute to the rising edge of the waveform, sincethe volume increase in the tissues would occur only after we have madeour cFMD measurement for a particular pulse. We thus believe that thecFMD metric is chiefly affected by dilation of the artery rather thansmaller colateral resistance vessels.

2.2. Study Protocol.

A typical study proceeds as follows:

(i) With the subject seated or supine, the cuff is placed around theupper arm.

(ii) Blood pressure is measured.

(iii) The cuff is inflated to a value Pm, which must be less than themean arterial pressure, for a period Tm=30 s. During this time interval,we measure and record the pressure fluctuations in the cuff. These dataconstitute a pre-stimulus baseline measurement.

(iv) The cuff is deflated.

Typically, Nb=3 baseline measurement series are obtained by repeatingSteps (iii)-(iv), with a waiting period of T_(w)=30 s betweeninflations. These rest periods allow restoration of venous return.

v) The stimulus is applied. This is either 1-5 min of cuff occlusion tosuprasystolic pressure Ps (for studies of endothelial function) or adose of sublingual NG (for studies of endothelium-independentvasodilation).

(vi) After T_(p)=45 s have elapsed following cuff release or drugadministration, a series of up to Nr=10 repeat measurement intervalsensue. In each interval, the cuff is inflated to P_(m) for T_(m)seconds, after which it is deflated for T_(w) seconds. This large numberof repeat measurements (Nr) is required only when one wishes to recordthe return of the vessel toward baseline.

(vii) Blood pressure is measured again to ensure it has not changedappreciably since step (ii).

(viii) Each post-stimulus response is then compared to the averagebaseline response, to yield the area-based cFMD metric (Equation 2)defined above. As is the objective in uFMD studies, we seek the value ofmaximal vasodilation within the response time course as a fraction ofthe baseline condition of the artery.

It is very important to ensure that P_(m) remains below the diastolicpressure throughout the entire study. Should P_(m) exceed the diastolicpressure, the artery will collapse during at least part of the cardiaccycle. This “clipping” of the pressure waveform will generally reducethe measured ΔP for each pulse. Since any subsequent increases in areachange will then be only partially reflected in the measurements, thequantity A_(r)/A_(b) may be underestimated.

Steps (ii) through (viii) can be completely automated and ensue withoutthe need for user intervention.

2.3. Device Prototypes

The following three prototypes implement the method. The successiveprototypes evolve not in terms of measurement quality (which is superiorin the first prototype), but in suitability for routine and home use.

2.3.1. Prototype I

FIG. 1 is a schematic of the first prototype. A rapid cuff inflator(E20, D.E. Hokanson, Inc., Bellevue, Wash.) is employed to set the cuffpressure to constant values for the occlusion and measurement intervals.This air source provides servo regulation that is too fast to allow itsdirect application to the cuff without attenuating the (desired) signaldue to the expansion of the arterial lumen. Consequently, we employ a 1m length of 2.79 mm-internal diameter intervening tubing, which servesas a pneumatic low pass filter.

The pressure in the cuff is measured using a pressure transducer. Thisprototype employs a Millar catheter pressure sensor (Mikro-tip, MillarInstruments, Houston, Tex.) for this purpose. The signal output of theMikro-tip system is amplified using an instrumentation amplifier (AD627,Analog Devices, Inc., Norwood Mass.) and a low-pass filter, with acut-off frequency of 25 Hz (8th order elliptic filter, LTC-1069-6,Linear Technology Corp., Milpitas, Calif.). It is then digitized at 1kHz using an A/D converter card (NI-6035, National Instruments, Austin,Tex.).

A PC controls the inflation and deflation of the cuff in accordance withthe protocol using a data line of its parallel printer port.

Prototype I is superior to Prototypes II and III, described below, interms of signal quality, since the unloading pressure is maintained at aconstant level throughout the measurement period.

2.3.2. Prototype II

To make a lower cost, more compact prototype, we replace thecontinuously regulated air source with an on-off pressure controlsystem, as shown in FIGS. 12 and 13. Inflation and deflation of the cuffare effected using a miniature diaphragm pump and solenoid valve(respectively, E161-11-050 and V2-20-5-PV-5-P88, Parker Hannifin Corp,Cleveland, Ohio). Cost is further reduced by employing a mass-marketsemiconductor pressure sensor (NPC-1210, GE Novasensor, Fremont,Calif.).

A script running on a laptop fully automates the measurement protocol.To modify the cuff pressure, the script sets a pressure-calibratedvoltage on a 12-bit digital-to-analog converter on the data acquisitioncard. A microcontroller (PIC12F675, MicroChip Technology, Inc., ChandlerAriz.) compares this voltage to the output voltage of the pressuresensor, and it actuates the pump and valve to maintain the desiredpressure within a specified tolerance.

A disadvantage of using an on-off control algorithm is that pressuretends to decrease during a measurement owing to displacement of the armtissue under the cuff. Frequent actuation of the pump to top-up air inthe cuff introduces artifacts into the acquired pulse waveform. In thedescription of Prototype III below, we show how the acquisition may bemodified to address this issue. Section 2.4 explains an alternativepost-hoc approach based on regression analysis.

2.3.3. Prototype III

A consumer-oriented electronic sphygmomanometer (Wireless Blood PressureMonitor, iHealth Lab Inc., Mountain View, Calif.) was modified by themanufacturer, under the supervision of our group, to implement theprotocol described in Section 2.2. The device operates in the samemanner as Prototype II.

The protocol parameters are set, and measurements are invoked, by acustom application (app) for Apple iOS handheld devices, includingiPhone and iPad (Apple Inc., Cupertino, Calif.). FIG. 19 shows thewireless cuff and the running app.

As shown in FIG. 6, a measurement interval of length T is divided intotwo segments, T1 and T2, such that T=T1+T2. The purpose of T1 is tostabilize the pressure close to the measurement pressure set-pointduring the period when tissue compression under the cuff leads to anatural pressure drop. Once the pressure has stabilized, T2 begins,during which no control of the pressure is exercised, or the criteriafor initiating pressure corrections are considerably relaxed.

Different pressure tolerances ΔP₁ and ΔP₂ may be applied to therespective time segments T₁ and T₂. During interval T_(n), adjustment ofthe pressure is only initiated when the cuff pressure P<P_(s)−ΔP_(n) orP>P_(s)+ΔP_(n). By setting, for example, ΔP₂>ΔP₁, it is possible toavoid unnecessary servoing during T₂ that may render measurement dataunusable. FIG. 20 provides an example of the specification of theseranges and the interpretation of these quantities.

2.4. Signal Processing

For the T_(m) second time record for measurement series i, p(t) isprocessed as follows:

(i) A 2-pole high-pass Butterworth filter with cutoff frequency of 0.5Hz is applied to remove the DC component of the cuff pressure signal,yielding p_(AC)(t).

(ii) A peak and foot detection algorithm identifies the individualpulses. Outliers in terms of pulse height, rise time, and period arediscarded.

(iii) For Prototype I, in which there is continuous pressure control,the remaining pulse heights are averaged to yield a value ΔP _(i) foreach measurement interval. For the other prototypes, which use on-offcontrol, linear regression is used to adjust the pulse heights to themean cuff pressure over all intervals i. The mean of the adjusted pulseheights for each i is then taken. This reduces bias introduced byvariations in unloading pressure that occur during each measurementinterval when on-off control is employed. These biases are introduced byshifting the operating point along the transmural pressure axis of FIG.16. Based on the behavior of these curves, it appears reasonable to fita linear model around an operating point close to 20 mmHg transmuralpressure.

(iv) The maximum of the cFMD metric in Equation 2, analogous to thatused for uFMD, expressed directly as a function of the measurement data,is calculated as

$\begin{matrix}{{{cFMD}_{{ma}\; x}\mspace{14mu} \%} = {\left\lbrack {\frac{\max_{N_{s} \geq k > N_{b}}\overset{\_}{\Delta \; P_{k}}}{{1/N_{b}}{\sum\limits_{n = 1}^{N_{b}}\overset{\_}{\Delta \; P_{n}}}} - 1} \right\rbrack \times 100}} & (4)\end{matrix}$

and reported, e.g., to the user. This value reflects the ratio betweenthe mean of all baseline measurement set means and the highest meanamong the post-stimulus measurement intervals. Where this metric appliesto general stimulus (e.g., reactive hyperemia or nitroglycerin), wedenote it cD_(max) %.

2.5. Evaluation in Human Subjects: Preliminary Studies

We seek first to establish whether the method:

-   -   (i) Is sensitive to smooth muscle relaxation due to sublingual        nitroglycerin.    -   (ii) Is sensitive to vasodilation following reactive hyperemia        in subjects with very low CVD risk.    -   (iii) Exhibits good repeatability.

Since the day-to-day FMD response is dependent on many factors (e.g.,food, medication, menstrual state and time-of-day), the consistency ofthe measurement method itself is best assessed via nitroglycerinstudies.

A total of three subjects are examined up to six times each for each ofthree stimuli:

-   -   (i) RH following 5 minutes of cuff occlusion (RH5);    -   (ii) 400 μg of sublingual nitroglycerin (NG); and    -   (iii) No stimulus (NS), equivalent to no cuffination, or zero        dose of drug.

Table 1, supra, provides details of the three subjects examined and thenumber of repeat tests performed for each stimulus. These subjects wereexamined at Lawrence Berkeley National Laboratory under an approvedhuman subjects protocol.

2.6. Evaluation in Human Subjects: Correlation Between cFMD and uFMD

While our small-sample preliminary studies can potentially provideevidence of the sensitivity and repeatability of the method, moreconvincing validation requires an adequately powered comparison of cFMDwith an accepted measure of FMD. We do this by comparing cFMD and uFMDmethods in the same subjects on the same day and at the same time ofday. We now describe the experimental design of this study.

2.6.1. Study Population.

We examined human volunteers currently involved in a study of theeffects of omega-3 fatty acid supplementation on vascular physiologicalparameters in patients with peripheral artery disease (PAD). Thesevolunteers consisted of subjects with known PAD and aged-matched,non-PAD controls. Most of the controls, however, were of advanced ageand had other cardiovascular disease. This population was chosen forconvenience and availability: inclusion of controls with a lower risk ofCVD would enable evaluation of the correlation between cFMD and uFMDover a wider range of endothelial competency. Since uFMD has highvariability, it is difficult to differentiate poor responders intomultiple tiers. The scatter of uFMD measurements alone can maskcorrelations for such groups. We proceeded with the studynotwithstanding this anticipated difficulty.

The characteristics of the subjects who participated in this study arelisted in Table 4. These subjects were examined at the San Francisco VAMedical Center, under approval from the relevant ethics board.

TABLE 4 Subject characteristics for cFMD/uFMD correlation study. Meanvalues are shown ± their standard deviations. Systolic HypersensitivesAll Excluded Number of subjects 27  16  # female 8 6 Age (years)  64.1 ±10.0 63.3 ± 10.1 Mass (kg)  86.0 ± 18.0 81.8 ± 17.9 BMI (kg/m²) 29.0 ±4.6 28.4 ± 4.8  # diabetic 7 4 # tobacco ever 17  9 # tobacco current 63 Systolic BP (mmHg) 144.8 ± 23.1 130.6 ± 7.5  Diastolic BP (mmHg) 87.3± 9.8 82.2 ± 4.9 

2.6.2. Ultrasound FMD Study Protocol

uFMD measurements were performed in accordance with currentlyrecommended guidelines and standards (Corretti et al. (2002) J. Am.Coll. Cardiol. 39: 257-265; Thijssen et al. (2011) Am I Physio.-HeartCirc. Physiol. 300(1): H2-H12) and as we describe in (Owens et al.(2009) J. Vasc. Surg. 50(5): 1063-1070). Before the study, subjects arerequired to fast for at least 8 hours and desist from nicotine productsfor at least 4 hours. A history of recent medications was recorded.Subjects rested for 10 minutes in a supine position in a darkened roomat 23° C. The subject's arm was then extended onto amovement-constraining pillow with the palmar aspect oriented anteriorly.A 5-cm-wide tourniquet blood pressure cuff was placed on the upper armdistal to the insertion of the deltoid. The length of the brachialartery was surveyed using B-mode ultrasound (Philips HD11, PhilipsHealthcare, Best, Netherlands) with a broadband linear array transducerwith a 3{12 MHz range (Philips L12-3) until a straight segment with avisible registration structure can be located. The probe was oriented sothat the artery was at least 3 cm below the surface of the skin, and thefocus is aligned with the deep boundary of the vessel. The protocolrequires that the boundary between the intima and lumen be clearlyvisible. Prior to cuffination, the baseline diameter of the vessel andblood flow velocity were recorded for 60 seconds usingelectrocardiogram-gated image capture software (Brachial Imager, MedicalImaging Applications LLC, Coralville, Iowa). Baseline blood flowvelocity was recorded for 60 s using an insonation angle of 60°. TheDoppler sample gate was positioned to cover the center, but not theedges, of the lumen. The probe remained in a fixed position betweenmeasurements. The blood pressure cuff was then inflated to the greaterof 250 mm Hg or 50 mm Hg above the subject's systolic blood pressure fora period of 5 minutes.

Recording of the B-mode images began 10 s prior to cuff release.Bloodflow velocity was assessed for a period of 30 seconds post-cuffrelease using the methods described above. B-mode images were recordeduntil 3 minutes post-cuff release. Analysis of the images was performedusing continuous edge-detection software (Brachial Analyzer, MedicalImaging Applications LLC). Baseline diameter was recorded as the mean of60 seconds of data. From recordings obtained during the reactivehyperemic phase, the exact moment of cuff release was determined.Hyperemia diameter was calculated using a pre-determined time window(55-65 s post-cuff release). uFMD % was calculated as:

${{{uFMD}\mspace{14mu} \%} = {100 \times \frac{d_{60S} - {\overset{\_}{d}}_{b}}{{\overset{\_}{d}}_{b}}}},$

where d_(60s) represents the diameter measured at 60 s after cuffrelease, and d_(b) is the average baseline diameter.

2.6.3. Sample Size Selection

We based our sample size on that recommended for uFMD, since ourpreliminary data suggested that the cFMD method is less variable andmuch more sensitive than uFMD.

Sample sizes of 20-30 per group have been previously used in uFMDstudies that attempt to compare endothelial function between two groups(Corretti et al. (2002) J. Am. Coll. Cardiol. 39: 257-65). With thissample size, the minimal statistically significant change that can bedetected with an intervention at this group size is an absolute changein FMD of 1.5% to 2% α=0.05, β=0.2 [power of 80%]).

The statistics obtained from 399 papers that appear in the meta-analysisof (Witte et al. (2005) J. Am. Coll. Cardiol. 45(12): 1987-1993) werealso useful for sample size selection. It is reasonable to expect thatthe measurement variance for a meta-analysis is higher than that forindividual laboratories and will consequently lead to an overestimate ofthe number of subjects required. Power analysis using the G*POWER 3.03software package (Erdfelder et al. (1996) Behav. Res. Meth. Inst. &Comp. 28: 1-11) for a power of 80% at a confidence level of 95% yieldeda sample size of 21 subjects per group to differentiate subjects in the1st and 3rd tertiles of Framingham risk, and 63 per group todifferentiate between the 1st and 2nd tertiles. Based on the literaturecited above, we chose a minimum group size of 21.

Since the purpose of this part of the study was to determine whethercFMD and uFMD are correlates, rather than investigate FMD underdifferent disease states, we combined the data from control and PADsubjects in one group.

3. Results

3.1. Preliminary Studies of cFMD

In Table 5 we calculate the maximum response for each stimulus andevaluate the statistical significance of the change relative to the nostimulus (NS) case (one-tail Student's t-test). Values of p<0:05 areconsidered significant.

TABLE 5 Statistical analysis of dilation response (cD_(max) %). Mean ±SEM of maximum p-value Stimulus response over all data sets cD_(max) %versus NS RH 1.51 ± 0.052 51% 1.19 × 10⁻⁵† NG 1.70 ± 0.036 70% 6.25 ×10⁻⁶† NS 1.01 ± 0.068 1% N/A †statistically significant SEM: standarderror of the mean

3.2. Flow-Mediated Dilation: Ultrasound-Versus Cuff-Based Measurements

FIG. 20 is a scatter plot that shows cFMD vs uFMD measurements for N=27subjects. The slope of the regression line indicates that cFMD is 346%more sensitive to the underlying stimulus than uFMD. When systolichypertensive subjects (those having systolic blood pressure greater than140 mmHg) are removed from the dataset, we found an increasedcorrelation, as shown in FIG. 21. (The rationale behind performing thisparticular analysis is based on the correlation between arterialstiffness and endothelial dysfunction observed in (Wallace et al. (2007)Hypertension, 50(1): 228-233). The relevance of those results to thepresent study is discussed below.)

Discussion.

A prudent first step in the evaluation of any new method or protocol forassessment of endothelial function is to establish sensitivity toendothelium-independent smooth muscle relaxation. By comparing theresponse of subjects to 400 μg and a zero dose of sublingual NG (nostimulus [NS]), we can establish whether the method is sensitive to thesmooth muscle relaxation that is the effect of endothelial stimulus.Smooth muscle relaxation and vasodilation are the end results of NOstimulus regardless of whether NO is endogenously generated orexogenously supplied.

The data shown in FIG. 10 demonstrate with great statistical certaintythat the proposed metric can detect changes due to NG vs. NS (+70%,p=6.25×10−6). Not only do the distributions for NG and NS responsesdiffer, but there is in fact no overlap of the distributions of thesedata within the time interval of maximum response, spanning from 5minutes to 15 minutes after the administration of the drug. We havepreviously determined that NG at this dose does not produce changes insystemic blood pressure that could confound these measurements (Maltzand Budinger (2005) Physiol. Meas. 26(3) 293-307). This is especiallyimportant in the case of the present method, as correct operationaccording to the arguments provided in Section 2.1 requires that bloodpressure remain constant between baseline and post-stimulus measurementintervals.

Since a 400 μg sublingual dose of NG is reported to elicit maximalsmooth muscle dilation (Feldman et al. (1979) Am. J. Cardiol. 43(1):91-97; Adams et al. (1998) J. Am. Coll. Cardiol. 32: 123-127) the nextstep was to determine whether RH following 5 minutes (RH5) of cuffocclusion produces a measurable change in the metric in individualsexpected to have sound endothelial function.

RH5 indeed produces a significant change vs NS (+51%, p=1.19×10−5). Inthe 4 minutes following cuff release, there is no overlap between theRH5 and NS distributions (during the window of maximum response) evidentin FIG. 10.

Table 5 summarizes the above findings.

These preliminary studies confirm that the method is sensitive tovasorelaxatory stimuli, but comparison with an established method isneeded to determine whether a proportional relationship exists betweenthe proposed and accepted metrics of endothelial function. FIG. 21displays a scatterplot of measurements from the established meathod ofuFMD and cuff FMD. We regard the correlation of r=0:55 observed in thedata depicted in FIG. 21 as moderate to strong, in view of the fact thatour study population has substantially poorer uFMD than would beexpected of a general population, and since our sample size limits us todifferentiation of the first and third tertiles of uFMD response. Ourpopulation sample was a convenience sample, with an over-representationof individuals with cardiac risk factors. The uFMD responses that weobserved in this study are typical of the first and second tertiles ofendothelial response for a larger sample of the general population. Weare thus not exploring the full natural \dynamic range” of FMD and thismakes it more difficult to observe stronger correlations.

Subjects with isolated systolic hypertension have been found to exhibitboth high aortic pulse wave velocity (arterial stiffness) and impairedFMD (Wallace et al. (2007) Hypertension, 50(1): 228-233). We thusperformed a sub-group analysis excluding subjects with systolicpressures above 140 mmHg, and found that the correlation between cFMDand uFMD increases to 0.82 (p<0.0002), as shown in FIG. 22. It ispossible that mechanical unloading of stiff arteries allows moreflow-mediated dilation to occur, since such arteries may not be asseverely restricted by their collagen framework when the wall is underless stress. (Models fit to in vivo measurements indicate that collagenfibers that act in parallel with the smooth muscle are increasinglyrecruited as transmural pressure rises (Bank et al. (1996) Circulation,94(12): 3263-3270)) If this is the case, uFMD may be systematicallyunderestimating FMD in these subjects. This contention is furthersupported by reported correlations between endothelium-dependent andendotheium-independent dilations (EDD and EID) (Adams et al. (1998) J.Am. Coll. Cardiol. 32: 123-127. In this large study of 800 subjects,Adams et al. found a correlation of 0.41 between EDD and EID. When thosesubjects at higher risk of atherosclerosis were removed (diabetics aswell as those with a history of tobacco smoking), the correlationcoefficient fell to 0.24. It is quite possible that the impaireddilation attributed to \smooth muscle dysfunction” (Id.) is in fact dueto an impaired ability of the vessel to dilate even when the smoothmuscle is relaxed.

It would be interesting to conduct a similar study to compare EDD andEID in the presence of mechanical unloading. Such studies may beconducted by measuring uFMD through a water-filled cuff. It is alsoimportant to confirm this finding by performing prospective studiesdesigned to validate this particular hypothesis on the sub-group.

Alternatively, if cFMD is overestimating dilation, the cFMD metric mayneed to be calibrated to systolic blood pressure in order to remove biasthat may occur in cases of subjects with systolic hypertension. Ourcurrent investigations are focused on understanding this phenomenon anddeveloping model-based calibration.

Our results show that the sensitivity of the method is between three andsix times greater than that of ultrasound-based imaging of arterialdiameter in response to both flow-mediated dilation and NG. Most of thissensitivity increase owes to our measurement of area rather thandiameter. As is often the case, a greater fundamental sensitivity to themeasured quantity makes it possible to use a simpler and lower-costmeasurement system. We have realized the measurement in a device that iscurrently marketed to the consumer at a price of $99.00 US.

In concordance with current recommendations (Thijssen et al. (2011) AmJ.

Physio.—Heart Circ. Physiol. 300(1): H2-H12), we believe measurements ofendothelial function in major arteries should ideally be based onNO-mediated FMD. In this sense, a limitation of the studies we performhere is that a single cuff is used for both measurement and occlusion.To assure that the dilation is purely NO-mediated requires a second cuffdistal to the measurement cuff. This is equivalent to the case ofwrist-occlusion in (Doshi et al. (2001) Clin. Sci. (Lond), 101:629-635), where eNOS inhibition abolishes, rather than merelyattenuates, FMD. The occlusion is then effected such that the measuredsegment of the artery is not subject to an ischemic stimulus during theocclusion interval. It is straightforward to modify the proposed methodand apparatus to realize a split- or separate-cuff design.

The combination of evidence and physical arguments presented heresuggests that cFMD and uFMD will remain correlated regardless of themethod of stimulus used.

While we have demonstrated that endothelial function may be assessedusing equipment of the same complexity as that used for blood pressuremeasurement, the time taken to acquire the data is considerably longer.In certain embodiments, the minimum time needed for a study is envisagedas equal to: baseline measurement time (15 s)+post-measurement recoverytime (30 s)+occlusion time+post-cuff-release time (60 s)+responsemeasurement time (15 s)=120 s+occlusion time. One way to shorten thestudy duration is to reduce the occlusion time. Corretti et al. (1995)Am. J. Physiol, 268: H1397-H1404, compared uFMD responses elicited byupper arm (proximal) occlusion times of 1, 3 and 5 minutes.Statistically significant responses were observed only in the case of5-minute occlusions. While the mean dilations for 1- and 3-minuteocclusions were substantial (respectively 2.1% and 7.8% vs 12.6% for5-minute occlusion), the data were extremely variable. There is thepossibility that owing to the sensitivity advantages of cFMD,measurements of the effects of a shorter occlusion might exhibit lowercoefficients-of-variation. A three minute occlusion would allowmeasurement of cFMD in 5 minutes, which is attractive in comparison toconventional protocols. Whether shortening the occlusion intervalchanges the physiological basis of the observed response can be assessedvia methods such as eNOS inhibition.

We believe the mass availability of a device for routine endothelialfunction assessment will prove clinically significant, since measurementof both acute and chronic changes in endothelial function could beaccomplished for the first time. There are compelling reasons to believethat knowledge of acute variation in endothelial function in anindividual is important. Since NO released by the endothelium is apotent inhibitor of the adhesion of platelets and leukocytes to theendothelial cell surface, and since adhesion of these cells is widelybelieved to be a necessary initiating event in atherogenesis (see, e.g.,Deanfield et al. (2005) J. Hypertens. 23: 7-17), it is reasonable toinfer that the proportion of time that the endothelium is dysfunctionalconstitutes an important indicator of disease risk. Just as dieters usea scale to measure body mass, and hypertensives use a home bloodpressure monitor, portable endothelial function monitors can provideindividuals with feedback regarding the impact of their lifestyle andmedications on arterial health.

Example 3 Method for Obtaining Model-Calibrated Arterial Endothelial andSmooth Muscle Function Measurements

A vast body of literature exists relating endothelial function, asmeasured via the conventional ultrasound imaging method, to variousstates of health and disease. Also, the influences of factors such asdiet, physical activity and medical interventions have been assessed viawhat we will refer to as uFMD (ultrasound-based flow mediated dilation)measurement methods. We have described a cuff-based method of assessingflow-mediated dilation (cFMD) that offers increased sensitivity bymeasuring cross sections area changes rather than diameter changes.While uFMD is based on changes in measures of an absolute quantity(arterial diameter), cFMD measures changes in a surrogate of vesselcross sectional area, namely the pressure in a cuff.

In order to relate cFMD to uFMD, and thus take full advantage of theexisting research on uFMD, and to more easily interpret study results,it is desirable to calibrate the two measurements.

In addition, in our studies of uFMD vs cFMD, we have found originalevidence that uFMD systematically underestimates smooth muscle responsein subjects with stiffer arteries. This means that clinicalinterventions aimed at improving endothelial or smooth muscle functionare not accurately assessed using the standard uFMD method. Our cFMDmethod, which performs the measurement on a mechanically unloadedmethod, is not only a more convenient and more sensitive method forthese subjects, but produces a less biased estimate. In these cases, acalibration method that can measure the true response, and relate thisto what would have been observed using uFMD is important forinterpretation of results.

To perform a calibration, it is convenient to have a suitable:

-   -   1. Measurement protocol; and    -   2. Mathematical model of the artery that may be based on a        combination of physical and empirical models.

Measurement Protocol

In order to perform a calibrated measurement, we adopt the followinggeneral protocol:

1. With the subject seated or supine, the cuff is placed around theupper arm (or other limb).

2. Blood pressure is measured.

3. The cuff is inflated to a value P_(m), which must be less than themean arterial pressure, for a period of time “Tm” (e.g., T_(m)=30 s).During this time interval, we measure and record the pressurefluctuations in the cuff. These data constitute a pre-stimulus baselinemeasurement.

4. The cuff is deflated. Typically multiple baseline measurement series(N_(b)) (e.g., N_(b)=3) series are obtained by repeating steps 3-4, witha waiting period (T_(w)) (e.g., T_(w)=30 s) between inflations. Theserest periods allow restoration of venous return circulation. The key toenabling calibration is that P_(m) is varied, either within each periodT_(m) and/or as a function of measurement interval index b. Typically,P_(m) would be varied in the range of 20-80 mmHg. Since the intention isto fit a parametric model, a model with K parameters requiresmeasurements of at least K different values. Typical values of K wouldbe 2, 3, 4 or 5.

5. The stimulus is applied. In certain illustrative embodiments, this iseither 1-5 min of cuff occlusion to suprasystolic pressure P_(s) (forstudies of endothelial function) or a dose of sublingual nitroglycerin(NG) (for studies of endothelium-independent vasodilation).

6. After time period T_(p) (e.g., Tp=45 s) has elapsed following cuffrelease or drug administration, a series of repeat measurment intervals“Nr” (e.g., up to Nr=10) ensue. In each interval, the cuff is inflatedto P_(m) for T_(m) seconds, after which it is deflated for T_(w)seconds. A large number of repeat measurements (e.g., N_(r)=10) isrequired only when one wishes to record the return of the vessel towardbaseline. Note that while it is desirable that the P_(m) used to measurethe post-stimulus responses be as close as possible to the P_(m) usedduring baseline, this is not essential, since it is possible toeffectively fit the parametric model even if these values differ or evendo not overlap.

7. Blood pressure can be measured again to ensure it has not changedappreciably since step (2).

8. The post-stimulus responses are then compared to the baselineresponses, to yield the area-based cFMD metric (Equation 1):

$\begin{matrix}{{{cFMD}\mspace{14mu} \%} = {\left\lbrack {\frac{A_{r}}{A_{b}} - 1} \right\rbrack \times 100.}} & (1)\end{matrix}$

where the the pre- and post-stimulus areas are denoted as A_(b) andA_(r), respectively. As is the objective in uFMD studies, the value ofmaximal vasodilation within the response time course as a fraction ofthe baseline condition of the artery is determined.

9. A calibration, such as that described in Section 3 below, can thenapplied to convert this value to a corresponding uFMD-equivalent value(by referencing the model to zero unloading pressure, i.e., P_(m)=0), orin order to formulate new FMD assessment metrics based directly on themodel parameters. Values of the parameters T_(m), P_(m), N_(b), N_(r),T_(w), and T_(p) given above are examples of typical values.

Model of Arterial Wall Used for Calibration and Formulation of NewMetrics

Background

In (Bank et al. (1999) Circulation, 100: 41-47), an empirical model isfit to the area versus transmural pressure curves of human arteries inbaseline and dilated states. FIG. 23 illustrates such a fittedarctangent model for an individual with a blood pressure of 122/77 mmHg.Conventional uFMD is measured at a transmural pressure equal to thesystolic blood pressure (no artificial mechanical unloading of thevessel wall is present). For example, to obtain uFMD from this figure,one would divide find the ratio between the rightmost value of the uppercurve A_(d)(122) and the rightmost value of the lower curve A_(b)(122)and take its square root to convert to a ratio of diameters:

${{uFMD}\mspace{14mu} \%} = {{\left( {{uFMD} - 1} \right) \times 100} = {{\left\lbrack {\sqrt{\frac{23.57}{16.99}} - 1} \right\rbrack \times 100} = {17.77{\%.}}}}$

It was a surprising discovery that that models such as these can be usedas a means to convert measurements obtained under mechanically unloadedconditions to one that would be obtained under the conventional unloadedcondition. The methods and devices described herein provide a protocoland measurement method for obtaining such curves that is much simpler,more sensitive, and economical than the methods used in (Bank et al.(1999) Circulation, 100: 41-47). Those methods involved either: 1)Ultrasound imaging through a water-filled cuff; or 2) Intra-arterialultrasound imaging (invasive) and application of pressure via externalair cuff. No attempt was made to convert measurements obtained to uFMDmeasurements.

The methods and devices described herein enable the measurement(typically by the averaging of many individual pulses) of the 2 samplesof each of the two curves used for the demonstration above. Once themodel is fit to the curves, these can be referenced to other measurementpressures than those used to acquire the data (including P_(m)=0, thecase for standard uFMD studies).

One Illustrative Embodiment of cFMD-uFMD Calibration

Let P_(D) and P_(S) represent the diastolic and systolic pressures,respectively. The cFMD measurement can be derived from four points onthe curve of FIG. 23, that are generally different from the two pointsused to calculate uFMD. These are A_(d)(P_(D)-P_(M)), andA_(d)(P_(S)-P_(M)) for the baseline curve and A_(d)(P_(S)-P_(M)), andA_(d)(P_(S)-P_(M)) for the curve for the dilated state. FIG. 23illustrates these four points using the symbols Δ and

depending on whether the measurements are obtained at diastole orsystole. The points obtained at diastole and systole are respectivelyindicated using the symbols Δ and

. The cuff pressure at which these measurements are obtained is 64 mmHgin this case. The transmural pressures are, respectively, 77−64=13 mmHgand 122−64=58 mmHg.

To relate uFMD and cFMD, consider, for example, the empirical3-parameter arctangent artery model employed in [1]:

$\begin{matrix}{{A\left( P_{tm} \right)} = {a\left\lbrack {\frac{1}{2} + {\frac{1}{\pi}{{atan}\left( \frac{P_{tm} - b}{c} \right)}}} \right\rbrack}} & (2)\end{matrix}$

As above, let A_(b) and A_(d) represent the baseline and dilatedarterial lumen cross sectional areas, respectively. uFMD is usuallydetermined at systole, where the transmural pressure is equal to thesystolic pressure, P_(tm)=P_(S). uFMD % is expressed in terms of (2) as:

$\begin{matrix}{{{uFMD}\mspace{14mu} \%} = {{\left( {{uFMD} - 1} \right) \times 100} = {\left\lbrack {\sqrt{\frac{A_{d}\left( P_{S} \right)}{A_{b}\left( P_{S} \right)}} - 1} \right\rbrack \times 100}}} & (3)\end{matrix}$

In a cFMD examination, P_(tm) is dependent on the systolic and diastolicblood pressures of the subject (P_(S) and P_(D), respectively), as wellas the pressure in the measurement cuff, P_(M):

TABLE 6 Parameters of arctangent model determined by fitting to datashown in FIG. 23. Parameter Baseline Dilated Ratio a 17.59 24.92 1.57 b−1.34 1.66 c 13.15 20.7

$\begin{matrix}{{{cFMD}\mspace{14mu} \%} = {{\left( {{cFMD} - 1} \right) \times 100} = {\left\lbrack {\frac{{A_{d}\left( {P_{S} - P_{M}} \right)} - {A_{d}\left( {P_{D} - P_{M}} \right)}}{{A_{b}\left( {P_{S} - P_{M}} \right)} - {A_{b}\left( {P_{D} - P_{M}} \right)}} - 1} \right\rbrack \times 100}}} & (4)\end{matrix}$

Applying the arctangent subtraction formula to (2), we have:

${{{A\left( P_{1} \right)} - {A\left( P_{2} \right)}} = {\frac{a}{\pi}{{atan}\left\lbrack \frac{\frac{1}{c}\left( {P_{1} - P_{2}} \right)}{1 + {\frac{1}{c}\left( {P_{1} - b} \right)\left( {P_{2} - b} \right)}} \right\rbrack}}},$

which gives:

${cFMD} = {\frac{a_{d}}{a_{b}} \times {\frac{{atan}\left\lbrack \frac{\frac{1}{c_{d}}\left( {P_{S} - P_{D}} \right)}{1 + {{c_{d}^{- 2}\left( {P_{S} - P_{M} - b_{d}} \right)}\left( {P_{D} - P_{M} - b_{d}} \right)}} \right\rbrack}{{atan}\left\lbrack \frac{\frac{1}{c_{b}}\left( {P_{S} - P_{D}} \right)}{1 + {{c_{b}^{- 2}\left( {P_{S} - P_{M} - b_{b}} \right)}\left( {P_{D} - P_{M} - b_{b}} \right)}} \right\rbrack}.}}$

Dimensional analysis of (2) indicates that a is in units of area, whichb and c are in units of pressure. Equation (6) may thus be interpretedas the product of an area ratio (approximately proportional to thesquare of uFMD) and a transmural pressure dependent “gain factor”.

It is instructive to examine the fitted values of a, b and c, whichappear in Table 6. The value of √{square root over (a_(d)/a_(b))} is1.25. Thi is similar to the ≈20% typical dilation due to nitroglycerinobserved at P_(t,)=P_(S).

When P_(tm)=b, which is a very small pressure (i.e., the artery isalmost completely unloaded), A=a/2. In this case, a_(d)/a_(b) is equalto uFMD² at large unlaoding pressure.

For the conventional case where uFMD is measured at systole with a fullyloaded artery, consider an artery with P_(s)=120. At baseline:

A _(b) =a _(b)(a tan((120+1.34)/13.15/π+½)=0.97a_(b).

After dilation we have:

A _(b) =a _(b)(a tan((120+1.66)/20.7/π+½)=0.94a_(b).

So, A_(d)/A/b=0.94/0.97×a_(d)/a_(b)=0.98 a_(d)/a_(b) which is very closeto (conventional, fully loaded) uFMD². It is therefore possible toconvert a cFMD measuremnt to uFMD by calculating the ‘gain factor” basedon P_(M), P_(S), P_(D) and this relationship between A_(d)/A_(b) anda_(d)/a_(b).

The above constitutes an apparently original factorization of a cFMDmetric into a product of an area parameter ratio (pressure-independent)and a factor that is dependent on both blood pressure and measurementpressure.

Generalizations

The above methods were described above with respect to measurement ofendothelial function. However, by changing the stimulus to nitroglycerin(for example), it is possible to assess basic smooth muscle function.Similarly, use of other stimuli (e.g., albuterol, acetylcholine,adrenergic agents, prostacyclins and endothelins) allows examination ofdifferent pathways involved in arterial dilation and constriction. Thearctangent model is only one of many possible models that describe thepressure-area relationship of the vessel.

Supporting Data and Arguments.

FIG. 24 is a scatter plot that shows cFMD vs uFMD measurements obtainedfor N=27 subjects. The characteristics of the subjects who participatedin this study are listed in Table 7. These subjects had cardiovascularrisk factor profiles that were less favorable than would be expected ofthe general population. This cohort is well-suited to demonstrate thedeficiencies of uFMD in cases where arterial stiffness may confoundresults.

TABLE 7 Subject characteristics for cFMD/uFMD correlation study. Meanvalues are shown ± their standard deviations. Systolic hypertensives Allexcluded Number of subjects 27  16  # female 8 6 Age (years)  64.1 ±10.0   63 ± 10.1 Mass (kg)  86.0 ± 18.0  81.8 ± 17.9 BMI (kg/m²) 29.0 ±4.6 28.4 ± 4.8 # diabetic 7 4 # tobacco ever 17  9 # tobacco current 6 3Systolic BP (mmHg) 144.8 ± 23.1 130.6 ± 7.5  Diastolic BP (mmHg) 87.3 ±9.8 82.2 ± 4.9When systolic hypertensive subjects (those having systolic bloodpressure greater than 140 mmHg) are removed from the dataset, we find anincreased correlation, as shown in FIG. 25. The rationale behindperforming this particular analysis is based on the correlation betweenarterial stiffness and endothelial dysfunction observed in (Wallace etal. (2007) Hypertension, 50(1): 228-233), and is discussed furtherbelow.

Discussion.

We regard the correlation of r=0.55 observed in the data for allsubjects depicted in FIG. 24 as moderate to strong, in view of the factthat our study population has substantially poorer uFMD than would beexpected of a general population. This correlation is substantiallyhigher than observed for EndoPAT, suggesting that the measurement has adifferent physiological basis.

Subjects with isolated systolic hypertension have been found to exhibitboth high aortic pulse wave velocity (arterial stiffness) and impairedFMD (Id.). We thus performed a sub-group analysis excluding subjectswith systolic pressures above 140 mmHg, and found that the correlationbetween cFMD and uFMD increases to 0.82 (p<0.0002), as shown in FIG. 25.It is possible that mechanical unloading of stiff arteries allows moreflow-mediated dilation to occur, since such arteries may not be asseverely restricted by their collagen framework when the wall is underless stress. Models fit to in vivo measurements indicate that collagenfibers that act in parallel with the smooth muscle are increasinglyrecruited as transmural pressure rises (Bank et al. (1996) Circulation,94(12): 3263-3270). If this is the case, uFMD may be systematicallyunderestimating FMD in these subjects. This contention is furthersupported by reported correlations between endothelium-dependent andendothelium-independent dilations (EDD and EID) (Adams et al. (1998) J.Am. Coll. Cardiol., 32: 123-127). In this large study of 800 subjects,Adams et al. found a correlation of 0.41 between EDD and EID. When thosesubjects at higher risk of atherosclerosis were removed (diabetics aswell as those with a history of tobacco smoking), the correlationcoefficient fell to 0.24. It is quite possible that the impaireddilation attributed to “smooth muscle dysfunction” (Id.) is in fact dueto an impaired ability of the vessel to dilate even when the smoothmuscle is relaxed. FIG. 26 shows the EDD-EID correlation observed inAdams et al. (1998) J. Am. Coll. Cardiol., 32: 123-127.

These results support the use of cFMD rather than uFMD for endothelialfunction assessment, and the desirability of being able to mutuallycalibrate the two measurements.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of assessing cardiovascular function ina mammal, said method comprising: a) applying external pressure P_(m) toan artery for a period of time T_(m), and determining, over the courseof one or more cardiac cycles in said period T_(m), changes in pressurein said artery resulting from cardiac activity of said mammal, or anartificially induced arterial pulse, to provide values for a baselineparameter related to endothelial function in said mammal, where saidbaseline parameter is provided for at least three different externalpressures (P_(m)) by varying the external pressure P_(m) during periodof time T_(m), and/or by measuring said baseline parameter over aplurality of time periods T_(m) having different external pressures(P_(m)), to provide a plurality of baseline values for said parameterrelated to cardiovascular function as a function of said externalpressure (P_(m)); b) applying a stimulus to said mammal; and c) applyingexternal pressure P_(m) to an said artery for a period of time T_(m),and determining over the course of one or more cardiac cycles in saidperiod T_(m), changes in pressure in said artery resulting from cardiacactivity of said mammal, or an artificially induced arterial pulse, toprovide values for a stimulus-effected parameter related to endothelialfunction in said mammal, where said stimulus-effected parameter isprovided for at least three different external pressures (P_(m)) byvarying the external pressure P_(m) during period of time T_(m), and/orby determining said parameter over a plurality of time periods T_(m)having different external pressures (P_(m)), to provide a plurality ofstimulus-effected values for said parameter related to cardiovascularfunction as a function of applied pressure (P_(m)); wherein saidbaseline values are determined from measurements made when said mammalis not substantially effected by said stimulus and differences betweensaid baseline values and said stimulus-effected values provide a measureof cardiovascular function in said mammal.
 2. The method of claim 1,wherein said method further comprises: determining the area, or ameasure proportional to the area, of the arterial lumen calculated fromthe baseline measured pressures as a function of transmural pressureP_(tm), where transmural pressure is determined as the differencebetween the baseline measured pressures and the external pressure P_(m)and fitting these data with a first non-linear model to provide a firstfunction describing baseline arterial lumen area as function oftransmural pressure; determining the area, or a measure proportional tothe area, of the arterial lumen calculated from the stimulus-effectedmeasured pressures as a function of transmural pressure P_(tm), wheretransmural pressure is determined as the difference between the measuredstimulus-effected pressures and the external pressure P_(m) and fittingthese data with a second non-linear model to provide a second functiondescribing stimulus-effected arterial lumen area as function oftransmural pressure; using said first function to calculate baselinearterial lumen area (A_(b)) (e.g., a measure proportional to the areaA_(b)) at a transmural pressure substantially equal to the systolicblood pressure; using said second function to calculatestimulus-effected arterial lumen area (A_(r))) (e.g., a measureproportional to the area A_(r)) at a transmural pressure substantiallyequal to the systolic blood pressure; and determining the equivalentultrasound-based FMD (uFMD) measure where uFMD is proportional to thesquare root of the ratio A_(r)/A_(b).
 3. The method of claim 2,comprising outputting said uFMD measure to a display, printer, orcomputer readable medium.
 4. The method of claim 2, wherein theequivalent ultrasound-based FMD is given asuFMD%=[√{square root over ((A _(r) /A _(b))}−1]×100
 5. The method ofclaim 2, wherein said first non-linear model and said second non-linearmodel are three parameter models.
 6. The method of claim 1, whereinbaseline values and stimulus effected values are determined for the samelimb or region.
 7. The method of claim 1, wherein baseline values aredetermined for a limb or region contralateral to a limb or region usedfor determining the stimulus effected values.
 8. The method of claim 7,wherein said contralateral limb or region is used for monitoring bloodpressure during measurement.
 9. The method of claim 8, wherein monitoredblood pressure is used to adjust the measurement pressure if bloodpressure changes during the measurement.
 10. The method of claim 1,wherein: said applying external pressure P_(m) to an said artery for aperiod of time T_(m) comprises applying a substantially constantexternal pressure P_(m) for a period of time T_(m); or said applyingexternal pressure P_(m) to an said artery for a period of time T_(m)comprises applying a continuously varying external pressure P_(m) for aperiod of time T_(m).
 11. The method of claim 1, wherein step (a) and/orstep (b) comprises providing said values for a baseline parameter and/orsaid values for a stimulus-effected parameter for at least 3 differentpressures (P_(m)), or for at least 4 different pressures (P_(m)), or forat least 5 different pressures (P_(m)), or for at least 6 differentpressures (P_(m)), or for at least 7 different pressures (P_(m)), or forat least 8 different pressures (P_(m)), or for at least 9 differentpressures (P_(m)), or for at least 10 different pressures (P_(m)), orduring a continuous pressure variation.
 12. The method of claim 1,wherein: said external pressure is less than the mean arterial pressure;or said external pressure is less than the mean diastolic pressure. 13.The method of claim 1, wherein: said baseline parameter and/or saidstimulus-effected parameter is provided for at least three differentexternal pressures (P_(m)) by varying the external pressure P_(m) duringperiod of time T_(m); or said baseline parameter and/or saidstimulus-effected parameter is provided for at least three differentexternal pressure points (P_(m)) by determining said baseline parameterover a plurality of time periods T_(m) having different externalpressure points (P_(m)).
 14. The method of claim 1, wherein during saidtime interval(s) said external pressure is maintained substantiallyconstant and held at different levels in different time intervals. 15.The method of claim 1, wherein said external pressure is adjusted todifferent levels during a single time period (T_(m)).
 16. The method ofclaim 1, wherein said providing baseline values and/or providingstimulus-effected values comprises: providing values for changes inpressure resulting from an artificially induced arterial pulse; orproviding values for changes in pressure resulting from cardiac activityof said mammal.
 17. The method of claim 1, wherein said substantiallyexternal pressure is below the average diastolic pressure measured forsaid subject or below an expected diastolic pressure for said subject.18. The method of claim 1, wherein the providing stimulus-effectedvalues is performed at least about 30 seconds after stimulus up to about5 minutes after said stimulus.
 19. The method of claim 1, wherein saidproviding, over the course of one or more cardiac cycles, changes inpressure in said cuff resulting from cardiac activity of said mammalcomprises determining the pressure in said cuff as a function of time.20. The method of claim 19, wherein: said providing comprisesintegrating the value of a pressure change over time (calculating thearea under a pressure/time curve) for one or for a plurality of cardiaccycles to determine an integrated pressure value; and/or said providingcomprises determining the maximum, or a certain percentile rank of thederivative of the pressure versus time wave form on the rising edge of apressure pulse for one or for a plurality of cardiac cycles to determinea compliance value.
 21. The method of claim 20, wherein said integratedpressure value and/or said compliance value is determined for a singlecardiac cycle.
 22. The method of claim 21, wherein said single cardiaccycle is a cardiac cycle selected for the maximum change in said valuein a plurality of cardiac cycles; or said single cardiac cycle is acardiac cycle selected for the maximum change in said value between abaseline measurement and a stimulus-effected measurement.
 23. The methodof claim 19, wherein said providing, over the course of one or morecardiac cycles, changes in pressure in said cuff resulting from cardiacactivity of said mammal comprises determining the pressure in said cuffas a function of time to provide a cardiac pulse waveform.
 24. Themethod of claim 23, wherein said providing, over the course of one ormore cardiac cycles, changes in pressure in said cuff resulting fromcardiac activity of said mammal comprises determining the pressure insaid cuff as a function of time to provide a cardiac pulse waveformwhile the cuff inflates and/or while said cuff deflates.
 25. The methodof claim 24, wherein the cardiac pulse waveforms are compared betweenbaseline and post-stimulus conditions by direct comparison of pulsecharacteristics at corresponding pressure levels; or the cardiac pulsewaveforms are compared between baseline and post-stimulus conditions byfirst fitting models to the set of baseline cardiac pulse waver formsand to the set of post-stimulus cardiac pulse waveforms and comparingparameters generated by the two models.
 26. The method of claim 24,wherein said external pressure is a pressure in excess of the peak ofthe cardiac pulse waveform (oscillometric waveform).
 27. The methodaccording of claim 24, wherein: the systolic BP and MAP are measured andthe diastolic blood pressure (DBP) is calculated using an oscillometricanalysis; or the systolic BP and MAP are measured and the DBP isdetermined by analyzing Korotkoff sounds obtained using an audio sensoror ultrasound probe.
 28. The method of claim 24, wherein measurements tocalculate cFMD and/or MAP, and/or DBP are obtained during inflation ordeflation of a sphygmomanometer cuff.
 29. The method according of claim24, wherein: systolic BP and/or diastolic BP is determined and for cFMDdetermination, comparison of pulse waveform characteristics betweenbaseline and post-stimulus intervals at corresponding pressures is acomparison made where the corresponding pressures are transmuralpressures calculated using the determined systolic and/or diastolic BPvalues; or comparison of pulse waveform characteristics between baselineand post-stimulus intervals at corresponding pressures is a comparisonmade based on the transmural pressure determined by the models.
 30. Themethod of claim 1, wherein applying the stimulus comprises restrictingflow of blood to the limb by occlusion of a blood vessel, oradministering a drug to said mammal or applying low intensity ultrasoundand/or acoustic/mechanical tissue vibration to said mammal.